ZA200106359B - Microbial process for preparing pravastatin. - Google Patents

Description

1 1 oo WO 00/46175 PCT/US00/02993 ] MICROBIAL FROCESS FOR PREPARING PRAVASTATIN

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of pravastatin, and particularly to a microbial process for the manufacture of pravastatin on an industrial scale.

BACKGROUND OF THE INVENTION

The highest risk factor of atherosclerosis and especially coronary occlusion is the high cholesterol level of the plasma. In the last two decades 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC.1.1.1.34) as the rate limiting key enzyme of the cholesterol biosynthesis was extensively examined. Pravastatin, a compound of Formula I, a WOH

NaOOC™ = sy

HO;

Jw

CH; i ’ CHs

R HO E] * a ’ -

OM and other related compounds (compactin, mevinolin, simvastatin) are the competitive inhibitors of the HMG-CoA reductase enzyme [A. Endo et al, J. Antibiot. 29, 1346-1348 (1976); A. Endo etal, FEBS Lett. 72, 323-326 (1976); CH. Kuo et al.. J. Org. Chem. 48, 1991 (1983)).

Pravastatin was first isolated by M. Tanaka et al. (unpublished results) from the urine of a dog during the examination of the compactin metabolism (Arai, M. et al, Sankyo

Kenkyusyo Nenpo, 40, 1-38, 1988). Currently pravastatin is a cholesterol lowering agent with the most advantageous action mechanism in the therapy. Its most important character is tissue selectivity, i.c., it inhibits the cholesterol synthesis at the two main sites of the cholesterogenesis, such as in the liver and in the small intestine, while in other organs the intracellular enzyme .

limiting effect is hardly detectable, At the same time the cholesterol biosynthesis limiting effect of mevinolin.and simyastatin is significant in most of the organs (T. Koga et al. Biochim. : .

Biophys. Acta, 1045, 115-120, 1990).

Pravastatin essentially differs in chemical structure from mevinolin and me simvastatin which have more lipophilic character. In the case of the latter compounds tue sub®ituent connected to the C-1 carbon atom of the hexahydr8ftaphthalene skeleton is ended in a 6-membered lactone ring, while in the case of pravastatin, instead of the lactone ring, the biologically active, opened dihydroxy acid sodium salt form is present. Another important structural difference is that instead of the methyl group of mevinolin and simvastatin at the C-6- position of the hexahydronaphthalene ring, a hydroxyl group can be found in pravastatin, which results in a further increase in its hydrophilic character.

As a result of the above structural differences pravastatin is able to penetrate through the lipophilic membrane of the peripheral cells only to a minimal extent (A.T.M,,

Serajuddin et al., J. Pharm. Sci. 80. 830-834. 1991).

Industrial production of pravastatin can be achieved by two fermentation processes. In the first, microbiological stage compactin is prepared, then in the course of a second fermentation the sodium salt of compactin acid as a substrate is converted to pravastatin by microbial hydroxylation at the 6f-position.

According to published patents. the microbial hydroxylation of compactin can be accomplished to various extents with mold species belonging to different genera, and with filamentous bacteria belonging to the Nocardia genus, with Actinomadura and Streptomyces . genera (Belgian patent specification No. 895090. Japanese patent specification No. 5,810,572,

US Patent Nos. 4,537,859 and 4,346,227 and published European patent application No. 0605230). The bioconversion of compactin substrate was published in a 500 pg/ml : concentration using filamentous molds such as Mucor hiemalis, Syncephalastrum nigricans,

Cunninghamella echinulata and in" 2000-4000 pg/ml with Nocardia, Actinomodura and -

Streptomyces strains belonging to the prokaryotes.

A general problem experienced in the cases of manufacturing the pravastatin with :

. filamentous molds is that due to the antifungal effect of compactin, the microorganisms are not able to tolerate the compactin substrate fed to the culture even at low concentrations (Serizawa et ; al., J. Antibiotics, 36, 887-891, 1983). The cell toxicity of this substrate was also observed in the hydroxylation with Strepromyces carbophilus. extensively studied by Japanese researchers (M.

Hosobuchi et al., Biotechnology and Bioengineering, 42, 815-820, 1993).

Japanese authors tried to improve the hydroxylating ability of the Streptomyces carbophilus strain with recombinant DNA techniques. A cytochrome P-450 monooxygenase system is needed for the hydroxylation of compactin (Matsuoka et al., Eur. J. Biochem. 184, 707- 713, 1989). However, according to the authors, in the bacterial cytochrome P-450 monooxygenase system not one but several proteins act in the electron transport, which aggravate the application of the DNA techniques. Development of a cost-effective microbiological hydroxylation method for the manufacture of pravastatin is an extremely difficult, complex task.

The aim of the present invention is to elaborate a new microbial process for the preparation of pravastatin from compactin in industrial scale, which would produce pravastatin at : more advantageous conditions than those previously known. During our research work, above all we tried to find a microorganism strain with a hydroxylase enzyme that can be adapted for the microbial transformation of compactin to pravastatin in a high concentration.

SUMMARY OF THE INVENTION

The present invention relates to a microbial process for the preparation of the compound of formula (I)

(3 WOH

NaOOC™ = gY . = - HO LA

CS J

- . CH; ~ p CH ee g hi -

RR © OT

HO Fa ZZ 3 (D from a substrate compound of formula (II). “OH

ROOC a

HO

O “Hl eo .

T H

CH; A CHa (ID wherein R stands for an alkali metal or ammonium ion. oo comprising the steps of (a) cultivating a strain of Mortierella maculata filamentous mold species able to 6B-hydroxylate a compound of formula (II) on a nutrient medium containing assimitable carbon- and nitrogen sources and mineral saits, (b) feeding the substrate to be transformed into the developed culture of Mortiere!!~ macuiatu, (c) fermenting the substrate until the end of bioconversion, (d) separating the compound of formula (I) from the culture broth, and (e) isolating the compound of formula (I).

The present invention also relates to a biologically pure culture of the Mortierella maculata n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial

} Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001266 and a biologically pure culture of its mutant, the Mortierella maculata n. sp. E-97/15/13 strain } deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest,

Hungary under accession number NCAIM(P)F 001267.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is an illustration of the physical characteristics of Mortierella maculata n. sp. E-97.

DETAILED DESCRIPTION OF THE INVENTION

In the course of our screening program, which covered about 5500 prokaryotic and eukaryotic strains, 23 microorganisms were selected, which were able to hydroxylate compactin in opposition. Among these strains a filamentous mold proved to be more appropriate for the production of pravastatin due to its higher resistance against compactin as compared to the strains known from published patents. According to the taxonomic investigation, this strain proved to be a new representative of the species belonging to the Mortierella genus (Mortierella maculata n. sp.). From the selected molds a new strain was isolated on the one hand by the application of the mutation-selection methods, and on the other hand by the induction of the hydroxylase enzyme of the strain. which one was able 10 hydroxyiate the compactin substrate to pravastatin in a higher concentration than published so far. As mutagenic agents, physical and chemical mutagens were applied (UV irradiation, methyl methane sulfonate, N-methyl-N'-nitro-

N-nitrosoguanidine). After the mutagenic treatments. in order to prepare haploid cells, the spore suspension was spread on benomyl-containing agar plates, then in order to induce the hydroxylase enzyme the developed colonies were inoculated onto 100 pg/ml 8-de-(2-methyl- butyryl)-compactin-contsining or compactin-containing agar plates. By the application of these methods a mutant strain was prepared from the new strain that is able to convert compactin to pravastatin to a significantly higher extent than the parent strain.

In the course of the optimizing experiments we determined the composition of the most beneficial inoculum, and the most advantageous bioconversion media for the compactin . hydroxylatioa.as well as the optimal method for the repeated feeding of eempactin in a high | . concentration.

Consequently, this invention is based on the recognition that the E-97 and E- 97/ 15/13 degienated strains of the isolated mold named Mortierella maculata, which were : mink deposited under Accession numbers of NCAIM(P)F 08T566 and NCAIM(P)F 001267 respectively, at the National Collection of Agricultural and Industrial Microorganisms (Department of Microbiology and Biotechnology. University of Horticulture and the Food

Industry Budapest), under appropriate fermentation conditions are able to manufacture pravastatin to a high extent, while the undesired related compounds such as the acid forms of 6a- hydroxy-compactin, 2a-hydroxy-compactin. 8-de-(2-methyl-butyryl)-compactin, 3a,58- dihydroxy-5,6-dihydro-isocompactin. 8a,B-hydroxy-compactin and the hydroxylated derivatives at positions 2 and 3 of the 2-methyl-butyrvl side chain of compactin are obtained only in small or - trace amounts during the bioconversion. Thus. these strains are especially appropriate for manufacturing pravastatin in an industrial scale.

Taking into account that the economical manufacture of the active ingredient on an industrial scale is a function of the compactin substrate concentration. it is important to have a strain that is able to tolerate high compactin and pravastatin concentrations. Consequently, a further important part of the invention is the recognition that the hydroxylating ability of the original mold isolate can be improved by the application of mutation-selection and enzyme induction methods and. furth=rmore, that by the development of an appropriate method for - substrate feeding the hydroxylation of large quantities of compactin to pravastatin can be executed in a single procedurc. 'n conclusion. the new mutant strain designated as Mortierella maculata n. sp. E-97/15/13 is especially appropriate for the manufacture of pravastatin.

Taxonomic features of the isolated new mold species comparing it to the most important diagnostic attributes of the known Mortierella species are summarized below. oo

Taxonomic description of the holotype strain Mortierella maculata nov. spec. E-97

: On starch-cascin-mait extract-agar media the aerial mycelium is well developed (more than 10 um thick covering layer over the substrate mycelium). At the beginning it appears - as a tightly woven white web of hyphae, in which later yellowish sporulating spots with a few mm diameter sparsely appear (new name "maculatus" refers to the above: spotted). This yellowish coloration can sometimes occupy the larger continuous surfaces of the aerial web. The color of the substrate mycelium is on Czapek-, bloody-Czapek-, tyrosine-, starch-casein-, malt extract-, etc. agar media mostly colorless or light yellowish. The color of the substrate mycelial web is light reddish on yeast extract-glucose-peptone medium. Production of diffusible and soluble pigments on the above listed media is not experienced, or only rarely an insignificant yellowish coloration occurs on these media. Colonies of strain E-97, due to their volatile oil production and similarly to many other species of Mortierella, (except to species of section 1sabellina), can exude a very characteristic strong scent.

Sporangiophores, designated by reference numerals 1-7 in Figure 1, frequently develop locally on the aerial hyphae (but less on the substrate ones) in great numbers at very different distances from each other. They are not branching, but are mostly straight or curved.

Their length is generally between 60-80pum. The starting point in the overwhelming majority of cases is a more or less short but strongly swollen hyphal section of the aerial web, from which they are separated by walls. Sporangiophores themselves can be also swollen (sometimes strongly), as shown by reference numeral 6. but in the direction of the sporangium they gradually narrow, from 5.0-9.0 pum to 1.0-2.0 pm. It is an important taxonomic character that below the sporangiophores they never broaden out (see reference numeral 8).

Sporangia are spherical; in some cases slightly flattened spheres. Their diameter is about 6.0-17.0 pm. relatively small compared to the measures of sporangia of other also exist. The spores 9 are cylindrical or less oval. Their size is 3.0-5.0 x 1.5-2.0 pm. Within the individual spores one or two small dark spherical oil-drops 18 may be present. Due to the very easy disintegration of the wall of sporangia, in wet surroundings the spores will quickly be scattered. After the disintegration of the sporangium. sometimes at the end of the sporangiospores, a fine pitchfork-like "collar" and a very short rudimental (and not typical) cotumella can be observed. Gemmae 15-28 that are spherical or cylindrical may occur onmost ‘ different diagnostic media. Usually the size is 10-25 pm. In the cultures chains of spherical gemmae 13, budding cells, intercalated gemmae 15-23, hyphal associations of particular spiral- growth of one hypha around the other 11, anastomotic-like structures and giant cells, etc. can also®s found. In the aerial mycelium also large (50-250 pm diameter) ei dense hyphal webs 14 can be seen but without the presence of detectable zygotes.

Cultures of strain E-97 are able to reduce nitrates to nitrites, do not hydrolyze starch, esculin, arginine or gelatine but hydrolyze Tween polysorbates and do not decompose paraffin hydrocarbons. The cultures of strain E-97 have urease activity show a good growth between pH 7.0 and 9.0 tolerate a maximum 2% NaCl. The effect of xanthine. hypoxanthine, lecithin. tyrosine and adenine are negative. A strong acid production of the cultures has been detected from glucose, fructose, glycerine and galactose, but very weak or no production from 3 xylose, arabinose, raffinose, sorbitol. inositol, inulin, etc. Weak growth is detected on pyruvate and acetate but no growth could be found with benzoate. salicylate, citrate, lactate. succinate, tartarate and malonate. A good growth was observed with glucose and fructose as sole carbon- sources in the medium. Utilization tests with xylose. arabinose. rhamrose. sucrose, raffinose, mannitol and inositol proved to be negative. The cultures do not decompose cellulose.

Systematic position: Strain E-97 belongs to the family Mortierellaceae and itis a typical member of the genus Mortierella: sporangia contain generally many spores, columeila is extr.mely reduced, gemmae are frequently present. the occurrence of zygotes has not been i) detected and the colonies exude a very characteristic strong scent. Within the genus Mortierella, strain E-97 is a typical representative of the "Section Alpina.” The latter can be characterized by very short non-branching sporangiophores (maximal length to 200 pm), and minute sporangia (Zycha, H. und Siepmann, R, Mucorales. Eine Beschreibung aller Gattungen und Ari... dieser

Pilzgruppe. D-3301 Lehre, Verl. von J. Cramer. 1969). Among the members of the section

Alpina, strain E-97 shows the greatest similarity to the species M. thaxteri Bjorling 1936 and M. renispora Dixon-Stewart 1932. However, the data in the Table 1 clearly show the differences in

: diagnostic properties of strain E-97 and of these two species.

Accordingly, as a new species herewith we introduce the strain under the name Mortierella maculata nov. spec.

E-97.

Table 1 - or ET] © Acor--arison of the holotype strain E-97 of Mortier~lla maculata n.sp. with the species M. renispora and M. thaxteri on the basis of key diagnostic properties ] Morticrella renispora Monierelia strain E-97 Mortierella thaxteni

Origin of _ - To Ordinary. however the | Laterally from (swollen or Laterally from swolier separated . sporangiophores ., . hyphac are wider than the normal) aerial hyphaé“of not segments of agial hyphae or not regular ones. laterally from separated sections of substrate separated segments of substrate hyphae broadened swollen hyphae. hyphae.

Shape and size of Gradually decreasing towards | Mostly straight or curved. not Length about 60-90um. Width at the sporangiophores the top from 10um to 3 um. branching. Width gradually starting point is about 3-7um. at the tip

Length is about 200 um. decreasing towards the tip: it is reduced to 1.5-2um. immediately from 3-9 um to 1.5-2.5um. under the sporangium broadened out

Length is about 60-80 um. At the tip never broadened. :

Shape and size of Colorless. diameter is 25um. Mostly spherical (6-17 um Spherical (12-20 um diam.). They sporangia diam.) but rarely less flattened. | contain many spores but on certain

Generally contain many media there is also only one spore- spores, rarcly one spore. bearing sporangra.

Wall (membrane) _| Spreading membrane. After Disintegrating. A pitchfork- Disintegrating. a minute backward - of the sporangia disintegration remains a like collar remains. bending cotlar remains. collar-like structure.

Shape and size of Roughly kidacy-shaped. Cylindrical. Length (3-5um) Ellipsoidal hyaline spores of 3.54 x the spores hyaline structures. sizes arc can doubly exceed the width 1.5-2 um) dimension. 2x4 um. (1.5-2 um).

Occur on the most different Frequent on very different Intercalary. oval gemmae (10-14um) in media media. mostly in the aerial the substrate mycelium. mycelium Spherical or clongased (10-25um).

Zygotes Frequem oa ail diagnostic ‘Net dewecied. Not observed. with the covering hyphae is about 500 um. without them about 30 um.

Dense foci of the Large densely woven hyphal In old cuhwures large (100-125 um hyphal web foci (50-250um) frequent but diam) yellowish-grey dense hyphal without zygotes. . webs. without zygote.

N

A

CT WO 00/46175 PCT/US00/02993 the acrial mycelium | web dense.

In the process for the preparation of pravastatin according to the present invention, preferably the culture of the mold strain designated as Mortierella maculata n. sp. E-97 or its mutant designated as E-97/15/13 is used. The selected strain is highly advantageous due to its fast growth.

As a carbon source it easily utilizes glucose, glycerine, fructose, or galactose. As a nitrogen source yeast extract, peptone, casein, meat extract, soybean meal, corn steep liquor, sodium nitrate, or ammonium sulfate can be used.

In the culture media used for the production of pravastatin besides the above carbon and nitrogen sources mineral salts. e.g., potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, trace elements (ferrous, manganous salts), amino acids and antifoaming agents, can be present.

According to a preferred embodiment of the present invention, the spore suspension having been prepared from the slant agar culture of the Mortierella maculata n. sp. designated as E- 97 strain or its mutant [NCAIM(P)F 001267] designated as E-97/15/13, is seeded into an inoculum medium; then 10% of the inoculum culture, which is cultivated for 3 days at about 25-30°C, preferably at about 24-28°C, most preferably at about 28°C, is transferred into the bioconversion medium. Then it is incubated for 4 days at about 25-28°C, preferably at about 28°C, then glucose and the sodium salt of compactin acid are fed into the culture. Depending on the concentration of the fed compactin substrate, the cultivation is continued for 2-12 days further under acrobic conditions, while the pH is maintained between 5.5 and 7.5. preferably at 7.0. The bioconversion is done under stirred and aerated conditions, when the air flow rate is 0.2 vvm, the spinning rate of the stirrer is 400/min.

In the course of the fermentation the bioconversion of compactin substrate was followed by a high pressure liquid chromatographic method (HPLC). According to this method, the sample of the broth is diluted twofold with methanol and centrifaged, and the supernatant is used for the HPLC analysis under the following parameters: Waters analytical HPLC equipment; column:

Nucleosil C,, 10 1am; detection wavelength: 238 nm; injection volume: 20 ul, flow rate: 1 mi/min; gradient elution is used, eluents: A=0.05% aqueous solution of phosphoric acid, B=acetonitrile.

Elution gradient: 0 vw 2 0] ow

IP IE 0 wm

Approximate retention times: pravastatin 8.6-9.0 min; compactin acid | 1.6-12.0 min; pravastatin lactone 15.0-15.5 min. compactin 16.5-17.0 min.

For the production of pravastatin the aqueous solution of the sodium salt of compactin acid is added at the 96th hour of the cultivation. For this procedure the substrate is prepared in solid form as follows. Compactin lactone is hydrolyzed in a 0.2M sodium hydroxide solution for 2 hours . at 40°C, then the pH of the reaction mixture is adjusted to 7.5 by hydrochloric acid and the neutralized solution is layered on a Diaion HP-20 adsorbent column; the sodium chloride formed during the neutralization is eliminated by aqueous washing of the column, and then the sodium salt of the compactin acid is eluted from the column by 50% aqueous acetone. Thereafter the eluate is distilled in vacuum and the aqueous residue is lyophilized. After neutralization the aqueous solution of the alkaline hydrolysate of compactin can also be directly used as substrate. In this case the compactin acid sodium salt content of the hydrolysate is measured by HPLC. and the solution is kept at 20°C until! being applied.

The higher the broth pH reached by the fourth day of the fermentation, the more - advantageous for the hydroxylation of the compactin substrate. Feeding of the compactin substrate is allowed to be started when the pH of the broth exceeds 6.3. At the 4th day of the fermentation as much of the sterile filtered aqueous solution of compactin acid sodium salt is added as needed to reach the 500 ug/ml concentration. Glucose is alsc fed to the culture from its 50% soluticx sterilized at 121°C for 25 minutes as follows: if the pH of the broth is higher than 6.7 value, 1% glucose is - added related to the volume of the broth, while if the pH is within the 6.3-6.7 range the quantity of the glucose fed is 0.5%. Compactin acid sodium salt is consumed from the broth after 24 hours, its

Co WO 00/46175 PCT/US00/02993 transformation is analyzed by HPLC measurement. In this case, for each ml of the broth another 500 ug of compactin is added. Besides the compactin substrate, glucose is also fed as described above. } Morphology of the 120 hour culture is characterized by the small pellet growth (diameter of the pellet; 0.5-3.0 mm). After 24 hours the second dose of substrate is also consumed from the broth, thus a further portion of compactin acid sodium salt producing a 500 g/ml concentration of it in the whole broth is added parallel with the glucose feeding dependent on the pH value of the broth. From the 4th day of the fermentation the substrate and the glucose feeding is repeated in daily frequency as it is written before until the 17th-18th day of the fermentation.

For the recovery of the product from the broth, it is advantageous to take into consideration the fact that during the bioconversion pravastatin is formed in its acidic form, thus it can be isolated from the filtrate of the broth by its adsorption on an anion exchange resin column.

For the isolation of the product it is advantageous to use a strongly basic anion exchange resin which is a polystyrene-divinylbenzene polymer carrying quaternary ammonium active groups. The product can be adsorbed directly from the filtrate of the broth by mixing the anion exchange resin being in hydroxyl form into it. The product being adsorbed on the ion exchange resin can be eluted from the column by acetic acid or a sodium chloride-containing acetone-water mixture, preferably 1% sodium chloride containing acetone-water (1:1) mixture. Pravastatin-containing fractions are combined and the acetone being in the eluate is distilled off in vacuum. The pH of the concentrate is adjusted with 15% sulfuric acid into the range of 3.5-4.0 and the aqueous solution is extracted by ethyl acetate. The ethyl acetate extract is washed with water and dried with anhydrous sodium sulphate. Then the lactone derivative is prepared from pravastatin. The lactone ring closure is carried out in dried ethyl acetate solution at room temperature, under continuous stirring by inducing the lactone formation with trifluoroacetic acid being present in catalytic quantity. The transformation procedure is checked by thin layer chromatographic analysis (TLC). After finishing the lactone formation the ethyl acetate solution is washed at first with 5% aqueous sodium hydrogen carbonate solution and then with water, then it is dried with anhydrous sodium sulfate and evaporated in vacoum. The evaporated residue is treated in acetone solution with charcoal, then evaporated again and recrystallized from a 1-4 carbon atom-containing aliphatic alcohol, preferably from ethanol. The evaporation residue of the recrystallization mother liquor is purified with silica gel column chromatography applying the mixture of ethyl acetate-n-hexane with gradually increasing ethyl acetate content as the eluent.

.

From the pravastatin lactone obtained after recrystallization and chromatographic purification pravastatin is prepared by hydrolysis at room temperature in acetone with equivalent quantity of sodium hydroxide. When the pravastatin sodium salt formation is completed, the reaction mixture is diluted with water and neutralized, and the acetone content is distilled in vacuum.

Pravastatin is adsorbed from the obtained aqueous residue on a Dia.on HP-20 resin-containing column, washed with deionized water and eluted from the column with apeacetone-deionized water mixture. Then the pravastatin containing fractions are combined, the acetone content is distilled off and after the lyophilization of aqueous residue pravastatin can be obtained in high purity, which can be recrystallized from an ethyl acetate-ethanol mixture.

In the course of the procedure the whole quantity of pravastatin can be adsorbed.

During the lactone closure of pravastatin 3a-hydroxy-iso-compactin and other by-products can also be formed. Although these latter reactions decrease the yield of the isolation but those compounds can be separated by the above-described purification method and consequently, pravastatin can be manufactured this way in a pharmaceutically acceptable quality. : :

After finishing the bioconversion pravastatin can be extracted either from the fermentation broth or from the filtrate obtained after the separation of the filamentous mold cells.

Filamentous mold cells can be eliminated either by filtration or centrifugation; however, it is advantageous especially on an industrial scale to make a whole broth extraction. Before extraction the pH of either the fermentation broth or the filtrate of the broth is adjusted to 3.5-3.7 with a mineral acid preferably with diluted sulfuric acid. The extraction is done with an ester of acetic acid and a 24 carbon atom containing aliphatic alcohol, preferably with ethyl acetate or isobutyl acetate. Extraction steps should be done very quickly in order to avoid the formation of the lactone derivative from pravastatin at acidic pH. )

From the organic solvent extract the pravastatin in acid form can be transferred 2s the sodium salt into the aqueous phase. For example, from an ethyl acetate extract pravastatin can be extracted by 1/10 and 1/20 volume ratio of 5% sodium hydrogen carbonate or weakly alkaline water (pH 7.5-8.0). It was found that pravastatin can be recovered in a pure form from the above-obtained alkaline aqueous extract by column chromatography with the application of a non-ionic adsorption ] resin. An advantageous method is to first remove the solvent dissolved in the aqueous phase by vacuum distillation from the alkaline aqueous extract, and then the aqueous extract is loaded on a oo WO 00/46175 PCT/US00/02993 : Diaion HP-20 column.

Pravastatin sodium salt being adsorbed on the column is purified by elution increasing gradually the acetone content of the aqueous solutions, then the pravastatin-containing main fractions are combined and concentrated in vacuum. The aqueous concentrate is purified further by chromatography on another Diaion HP-20 column, obtaining an eluate containing pure pravastatin, from which after clarification with charcoal and lyophilization pravastatin can be obtained in a pharmaceutically acceptable quality.

This isolation procedure consists of fewer stages than the previous one, since the lactone formation of pravastatin and its hydrolysis are not involved in the procedure. During the isolation pravastatin is exposed to acidic condition for only a limited time, under which it is less stable than in neutral or alkaline solutions, consequently, during this isolation procedure artefacts are practically not formed.

Furthermore, it was found that the chromatography on Sephadex LH-20 Dextran gel (hydroxypropylated derivative) is advantageously used for purifying pravastatin. By application of this method pravastatin exceeding the purity of 99.5% (measured by HPLC) can be produced.

In the course of our experiments it has been recognized that from the organic solvent extract, preferably from the ethyl acetate or isobutyl acetate extract of the broth or the broth nitrate of the filamentous mold or the filamentous bacteria strains among them the Mortierella maculata n. sp. strain able to 6B-hydroxylate a compound of general formula (II), pravastatin can be precipitated as a crystalline salt with secondary amines. Further it was found that for the salt formation several : secondary amines containing alkyl. cycloalkyl-, aralkyl- or aryl-substituents are appropriate.

Expediently non-toxic secondary amines were selected among them, e_g., dioctylamine, dicyclohexylamine, dibenzylamine. The isolation of the organic secondary amine salt intermediates, ¢.g., the dibenzylamine salt was carried out by adding dibenzylamine in 1.5 equivalent quantity related to the pravastatin content of the extract, then the extract is concentrated by vacuum distillation to 5% of its origimal vole, then another quantity of dibenzylsmine is added into the concentrate in 0.2 equivalent ratio. The crystalline dibenzylamine salt is precipitated from the concentrate. The crystalline crude product is filtered and dried in vacuam. Then it is clarified with charcoal and recrystallized in acetone.

In the procedure mentioned earlier in which the organic solvent extraction and the reextraction at alkaline pH are involved, the isolation method based on the secondary amine salt fouwation can be used also for the replacement of the column chromatographic purification. In this case it is advantageous to precipitate the pravastatir dibenzylamine salt from the isobutyl acetate extract obtained after the acidification of the alkaline aqueous extract.

Pravastatin organic secondary amine salts can be transformed to pravastatin by sodium hydroxide or a sodium alkoxide, preferably sodium epoxide. CL

The transformation is detailed in the case of pravastatin dibenzylamine salt. The recrystallized dibenzylamine salt is suspended in an isobutyl acetate-water mixture, then equivalent quantity of sodium hydroxide is added in aqueous solution to the suspension by maintaining under stirring the pH in the range of 8.0-8.5. After disappearance of the suspension the phases are separated and the pravastatin-containing aqueous solution is washed twice with isobutvl acetate. The aqueous solution is ~'7r¥i2d with activated carbon and lyophilized yielding pravastatin in a pharmaceutically acceptable quality.

One preferred method for the transformation of pravastatin dibenzylamine salt to i pravastatin is to suspend the recrystallized dibenzylamine salt in ethanol. then equivalent quantity or small excess of sodium ethoxide is added under stirring to the suspension, then the reaction mixture is concentrated in vacuum and by adding acetone the pravastatin is precipitated in crystalline form from the concentrate.

Another preferred method for the transformation of pravastatin dibenzylamine salt to pravastatin is to dissolve the recrystallized dibenzylamine salt in ethyl acetate-ethanol mixture and by adding equivalent quantity or small excess of sodium hydroxide in ethanol to the solution pravastatin is precipitated.

The isolation of pravastatin via a secondary amine salt intermediate is a simpler ~ formed. and the separation of pravastatin from the by-products of the bioconversion and from the various metabolic products biosynthesized by the hydroxylating microorganism can be solved S.- without the application of any chromatographic methods. ©... ... The structures of pravastatin, pravastatin lactone and the isolated secondary amine salts of pravastatin have been proven by UV, IR, 'H-NMR, C-NMR and mass spectroscopic methods.

ol WO 00/46175 PCT/US00/02993 } EXAMPLES

The invention will be more fully described and understood with reference to the } following examples, which are given by way of illustration and are not intended to limit the scope of the invention in any way.

Example 1

A spore suspension was prepared with 5 ml of a 0.9% sodium chloride solution obtained from a 7-10 day old, malt extract-yeast extract agar slant culture of Mortierella maculata nov. spec. E-97 [NCAIM(P)F 001266] strain able to 63-hydroxylate compactin and the suspension was used to inoculate 100 ml inoculum medium PI sterilized in a 500 ml Erlenmeyer flask.

Composition of the medium PI: glucose 50g soybean meal 20g in 1000 ml tap water.

Before the sterilization the pH of the medium was adjusted to 7.0, then it was sterilized at 121°C for 25 min. The culture was shaken on a rotary shaker (250 rpm, 2.5 cm amplitude) for 3 days at 28°C, then 10 ml of the obtained culture was transferred into 100-100 ml bioconversion media MU/4 sterilized in 500 ml Erlenmeyer flask for 25 min at 121°C.

Composition of the medium MU/4: glucose 40g soybean meal 20g cascin-peptone lg 3 2g potassium dihydrogen phosphate 0.5¢ im 1000 ml tap-water.

Before the sterilization the pH of the medium was adjusted to 7.0, then it was sterilized at 121°C for 25 min.

Flasks were shaken on a rotary shaker (250 rpm, 2.5 cm amplitude) for 4 days at 25°C,

then 50-50 mg compactin substrate (compactin acid scdium salt) was added in sterile-filtered aqueous form into the cultures, then the cultivation was continued. Similarly, at the 5th day.another 50-50 mg : compactin acid sodium salt was added into. the mold culiures, and the fermentation was continued for a further 24 hours. The pravastatin content of the broth was determined by HPLC. The fermentation a. Was continued for 168 hours. At the end of the bioconversion the average pravastatin concentration of 1% fermentation broth was 620 ug/ml. o-oo

Example 2

In a laboratory scale fermenter with 5 liters working volume a MU/S bioconversion culture medium is prepared, the components of the culture medium are added corresponding to 5 liters. volume but it was loaded up only to 4.5 liters. then it was sterilized for 45 min at 121°C and seeded with 500 ml of the inoculum culture made according to the Example 1.

Composition of medium MU/8: glucose 20g glycerine 20g soybean meal 20g peptone Sg potassium dihydrogen phosphate 0.5g polypropylenegivcol 2000 lg in 1000 ml tap water.

Before sterilization the pH of the medium was adjusted to 7.0 value.

The fermentation was carried out at 28°C, with a stirring rate of 400 rpm and with an = aeration rate from bottom direction 60 liters/hour for 4 days. At the 2nd day after the transfer the culture started to foam heavily, which ~an be decreased by the addition of further polypropyleneglycol 2000. At the beginning of the fermentation (16-20 hours) the pH decreased a from the initial value of 6.5 10 5.0-5.5, then from the 3rd day it started to increase and reached 6.3-7.5 by the 4th day. The feeding of the compactin substrate is allowed to be started if the pH of the broth: is above 6.3. At the 4th day of the fermentation 2.5 g compactin substrate is added in sterile filtered

: aqueous solution. Calculated for the volume of the broth 0.5-1.0% glucose was added into the culture depending on the pH in the form of 50% solution sterilized at 121°C for 25 minutes in parallel with the substrate feeding. After 24 hours the compactin substrate is consumed from the culture, which is detected by HPLC from the samples taken from the fermenter. In this case another 2.5 g compactin substrate and glucose were added as described above, and the bioconversion was continued for 24 hours further when the substrate was converted to pravastatin.

After finishing the fermentation, 5.1 liters broth containing 630 ng/ml pravastatin were filtered on a filter cloth. Two liters water were added to the separated mycelium, then the mycelium suspension was stirred for one hour and filtered. These two filtrates were combined and passed through with a flow rate of 500 ml/hour on a column containing 138 g (250 ml) Dowex Al 400 (OH) resin (diameter of the column 3.4 cm, height of the resin bed: 28 cm), then the resin bed was washed with 300 ml deionized water. Subsequently, the elution from the resin was carried out by 1 liter acetone-water (1:1) mixture containing 10 g sodium chloride. The volume of the fractions was 100 ml each. The eluate was analyzed by the following thin layer chromatographic (TLC) method: adsorbent: Kieseigel 60 F 254 DC (Merck) aluminum foil; developing solvent: acetone-benzene-acetic acid (50:50:3) mixture; detection: with phosphomolybdic acid reagent. The R¢ value of pravastatin is 0.5. Fractions containing the product were combined and the acetone was distilled off in vacuum.

The pH of the 400 mi concentrate was adjusted to 3.5-4.0 by 15% sulfuric acid, then it was extracted three times by 150 ml ethyl acetate. The ethyl acetate extracts were combined and dried with anhydrous sodium sulfate. Subsequently, pravastatin lactone was prepared from pravastatin acid by adding at room temperature under continuous stirring trifluoroacetic acid in catalytic amount. The formation of pravastatin lactone was controlled by TLC (the R; value of pravastatin lactone in the above TLC system is 0.7). Afier the completion of the lactone formation, the ethyl acetate was washed with 2x50 ml 5% agueous sodinm hydrogen carbonate solution, then washed with 50 mi water, dried with aslrydrous sodium sulfate and evaporated in vacuum. The evaporation residue obtained in a quantity of 3 g was dissolved in 100 mi acetone and clarified with 0.3 g charcoal. Then the charcoal was filtered off and the acetone was evaporated in vacuum. The crude product obtained was crystallized from 20 mi ethanol. Precipitated crystalline pravastatin lactone was filtered off, and washed on the filter with 30 ml n-hexane and dried at room temperature in vacuum. In this way 1.5 g chromatographically pure pravastatin lactone was obtained. Meltint noint 140-142°C, [alo = +194° (¢=0.5, methanol). The mother liquor of the crystallization was evaporated in vacuum and 1.2 g evaporation residue is obtained, which was chromatographed on 24 g Kieselgel 60 adsorbent containing column (diametcr of the column: 1.6 cm, height of the bed: 20 cm). The crude product dissolved in'5%nl benzene was layered on the column. For elution mixtids of ethyl acetate-n-hexane were used in which the ethyl acetate content was gradually increased. Pravastatin lactone can be eluted from the column with the mixture of 60% ethyl acetate - 40% n-hexane. Fractions were controlled by TLC using the mixture of ethyl acetate-n-hexane (9:1) as the developing solvent. The pravastatin lactone-containing fractions were combined and evaporated in vacuum. According to this method 0.3 g pure product is obtained. its quality identical with that of the pravastatin lactone obtained by crystallization.

The two pravastatin lactone batches were combined and the sodium salt was prepared ] according to the following method: 1.8 g pravastatin lactone was dissolved in 20 ml acetone and under stirring 4.5 ml of 1M aqueous sodium hydroxide was added. then the solution was stirred for half an hour at rooin temperature. When the salt formation was completed, 20 ml water was added into the mixture and the solution was neutralized. then the acetone was evaporated in vacuum. The aqueous concentrate was chromatographed on a column filled with 150 ml Diaion HP 20 resin (diameter of the column: 2.6 cm. height of the bed: 30 cm). As the eluting agent mixtures uf acetone- deionized water were used. where the concentration of the acetone was increased in 5% steps.

Pravastatin can be eluted from the column by a 15% acetone containing acetone-deionized water . mixture. Fractions were analyzed by TLC. Fractions containing the product are combined and acetone was evaporated in vacuum. By lyonhilization of the aqueous residue 1.3 g pravastatin was obtained. The chromatographically pure product was crystallized from a mixture of ethanol and ethyl acetate.

Melting point: 170-173°C (decomp.) [e]° =+156°, (c=0.5, in water).

Ultraviolet absorption spectrum (20 ug/ml. in methanol): A, =231, 237,245 nm

: (log £-4.263; 4.311; 4.136)

Infrared absorption spectrum (KBr): vOH 3415, uCH 2965, vC=0 1730, vCOO" 1575 cm. : 'H-NMR spectrum (D,O, 8, ppm): 0.86, d, 3H (2-CH,); 5.92, dd, J=10.0 and 5.4 Hz, 1H (3-H); 5.99, d, J=10.0 Hz, IH (4-H); 5.52, br 1H (5-H); 4.24, m 1H (6-H); 5.34, br, IH (8-H); 4.06, m, 1H (B-H), 3.65, m, 1H (6-H); 1.05, d, 3H (2'-CH;); 0.82, t, 3H (4'-H;). 3C-NMR spectrum (D,0, 8, ppm): 15.3, q (2-CH,); 139.5, d (C-3); 129.5, d, (C-4); 138.1, s (C-4a), 127.7, d (C-5); 66.6, d (C-6); 70.1, d (C-8); 182.6 s (COO); 72.6. d (C-p); 73.0, d (C-d); 182.0, s (C- 1) 18.8; q (2'-CH;); 13.7, q (C4).

Positive FAB mass spectrum (characteristic ions): [M+Na]" 469; [M+H]" 447.

Negative FAB mass spectrum (characteristic ions): [M-H} 445, [M-Na] 423, nv/z 101 [2-methyl-butyric acid-H]J.

Example 3

In a laboratory scale fermenter with 5 liters working volume, bioconversion culture medium MU/4 was prepared as described in Example 1, although it was loaded up to 4.5 liters, the composition of the culture medium was calculated to S liters. Then it was sterilized for 45 min at 121°C and inoculated with 500 ml of the inoculum culture made according to Example 1. The fermentation was carried out at 25°C by the application of a stirring rate of 300 rpm and an aeration rate of 50 liters/hour for 4 days. After 5 g compactin substrate feeding to the cutture the bioconversion was carried out according to the Exampie 2.

After finishing the bioconversion, the 4.9 liters broth, which contained 660 g/ml pravastatin, was filtered and the separated mycelium was washed by suspension in 2x1 liter deionized water. The pH of the combined 5.6 liters filtrase of the broth was adjusted by 20% sulfuric acid to 3.5-3.7, then the acidic filtrate was stirred with 2750 ml ethyl acetate for 30 min. Subsequently, the phases arc seperated. The agueoes phase was extracted again with 2x 1375 ml ethyl acetate. 470 mi deionized water was added to the combined 4740 ml ethyl acetate extract, then the pH of the aqueous ethyl acetate mixture was adjusted to 7.5-8.0 by 1M sodium hydroxide. After 20 min stirring the :

phases were separated, then the ethyl acetate extract was extracted with 2x235 ml deionized water as . described ahave. Then the combined weakly alkaline aqueous solution of 1080 ml volume was concentrated in vacuum to 280 ml volume. The concentrated aqueous solution was layered ona chromatographic column (ratio of height:diameter = 6.5) filled with 280 ml Diaion HP-20 (Mitsubishi<Co., Japan) non-ionic resin. The adsorption on the column was carried out with a flow =F rate of 250-300 hil/hour, then the column was washed With ‘840 ml deionized water. ‘Subsequently, the column was eluted in the following order with 860 mi 5%, 1000 ml 10%, 500 ml 15% and 500 ml 20% acetone-containing water. In the course of the elution 50 ml fractions were collected. which were analyzed by the TLC method given in the Example 2. Fractions containing pravastatin as the main component were combined and the obtained solution was concentrated in vacuum to 260 ml volume. The concentrated aqueous solution was chromatographed on a column containing Diaion

HP-20 resin in 260 ml volume. After the adsorption of pravastatin the column was washed with 790 ml deionized water, then eluted with aqueous acetone solutions in 260-260 ml portions gradually - increasing the acetone content as follows: 2.5.5.0. 7.5, 10.0, 12.5. 15.0 and 20.0%. In the course of the column chromatography 25 ml fractions were collected. and the pravastatin content of the fractions was analyzed as given before. Fractions containing pravastatin as the single component by

TLC were combined and evaporated in vacuum. Subsequently, 0.3 g charcoal was added to the concentrated aqueous solution (about 30 ml) and pravastatin was clarified at room temperature for 30 min. Then the charcoal was removed by filtration from the solution and the filtrate was lyophilized.

In this way 1.62 g pravastatin was obtained in lyophilized form.

Example 4

From the sian: .lture of the Mortierella maculata nov. spec. E-97 INCAIM(P)F 001266] strain cultivated for 10-12 days, a spore suspension was prepared with 5 ml sterile 0.9% sodium chloride solution, and this suspension was used to inoculate 500 ml VHIG inoculum medium being sterilized in 3000 mi Erlenmeyer flask.

Composition of the medium VHIG: glucose 30g

NE

CT WO 00146175 PCT/US00/02993 meat extract 8g yeast extract ig

Tween-80 (polyoxyethylene (20) sorbitan monooleate) 0.5g in 1000 ml tap water.

Before the sterilization the pH of the medium was adjusted to 7.0 and the sterilization was carried out at 121°C for 25 min. The culture was cultivated for 3 days on a rotary shaker (250 rpm, amplitude 2.5 cm), then the obtained inoculum culture was used to inoculate a laboratory scale fermenter containing bioconversion culture medium PX in 5 liters working volume.

Composition of the medium PK: glucose 40g peptone 5g soybean meal 20g

K-HPO, 2g

KH,PO, lg

NaNO, 2g

KCl 0.5g in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 7.0. After the inoculation, cultivation, the substrate feeding and bioconversion were carried out according to Example 2, then the pravastatin was isolated from the broth in which its concentration was 650 ug/ml at the end of the fermentation.

Finishing the fermentation. the pH of the 4.9 liters broth comaining 650 ug/ml pravastatin was adjusted under continuous stirring with 2M sodium hydroxide to 9.5-10.0, then after one hour stirring the pH is adjusted to 3.5-3.7 with 20% sulfuric acid. Subsequently, the acidic solution was extracted with 2.45 liters ethyl acetate. The phases are separated, and with centrifugation a clear extract was prepared from the emulsified organic phase. The broth was extracted again with 2x1.22 liters cthyl acetate by the method given above. The ethyl acetate extracts were combined and 0.4 liters deionized water were added, then the pH of the mixture was adjusted to 8.0-8.5 with 1M sodium hydroxide. Phases were seperated, and the ethyl acetate phase was extracted with 2x0.2 liters deionized water of pH 8.0-8.5 as given above. The pH of the combined pravastatin containing weakly alkaline aqueous solution was adjusted under stirring with a 20% sulfuric acid solution to 3.5-3.7. The acidic solution obtained was extracted with 4x0.2 liters ethyl acetate. The combined ethyl acetate extracts are washed with 2x0.2 liters deionized water, then 150 mole% dibenzylamine -- calculated for the pravastatin content measured by HPLC -- was added into the ethyl acetate solution. The ethyl acetate solution was concentrated in vacuum to 0.2 liters volume. Further

Mole% dibenzylamine was added to the concentrate obtained, and the precipitated solution was kept overnight at 0-5°C. The precipitated pravastatin dibenzylamine salt was filtered, then the precipitate was washed on the filter with cold ethyl acetate and then two times with n-hexane, and finally it is dried in vacuum at 40-50°C. The crude product obtained (3.9 g) was dissolved in 100 ml! methanol at room temperature, then the solution was clarified by 0.45 g charcoal. Thereafter the methyl alcoholic filtrate is concentrated in vacuum. The evaporated residue was dissolved in 120 ml acetone at an external temperature of 62-66°C, then the solution was cooled to room temperature.

Subsequently, the recrystallization was continued overnight at 0-5°C. Precipitated crystals were ) filtered, then the crystals were washed on the filter two times with cold acetone and two times with-n- hexane. The recrystallized pravastatin dibenzylamine salt was suspended in the mixture of 160 ml isobutyl acetate and 80 mi deionized water. Subsequently, sodium hydroxide was added in an equivalent amount into the suspension under stirring. After the disappearance of the suspension the phases were separated and the pravastatin containing aqueous soiution was washed with 2x30 ml isobutyl acetate. The aqueous solution obtained was clarified with charcoal. Then the aqueous filtrate was concentrated to about 20 ml volume. The aqueous solution obtained was loaded on a chromatographic column (height:diameter = 22) filled with 0.4 liters Sephadex LH-20 gel (supplier: R

Pharmacia, Sweden). In the course of the chromatography deionized water was used as the eluent, and 20 m! fractions were collected. Fractions were analyzed by TLC, then those containing pravastatin also by HPLC using the methods described above. Fractions containing pure pravastatin . were combined and lyophilized. In this way 1.75 g pravastatin was obtained, the pu...y of which is higher than 99.5% by HPLC. : 3

Example 5

. A spore suspension was prepared from the slant culture of the Mortierella maculata n. spec. E-97 [NCAIM(P)F 001266] strain cultivated for 10-12 days with 5 ml sterile 0.9% sodium . chloride solution, and then 500 ml inoculum medium was inoculated with it as described in Example 4. In a laboratory scale fermenter with 5 liters working volume bioconversion culture medium PC/4 is sterilized for 45 min at 121°C and then inoculated with the seed culture.

Composition of the medium PC/4: malt extract 5.0% soybean meal 1.0% peptone 1.0% corn steep liquor 1.0%

MgSO, x 7 H,0 0.1% in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 7.0. After the inoculation, the cultivation and substrate feeding were carried out according to the Example 2, and then 5.1 liters broth with a concentration of 610 ug/ml pravastatin was obtained.

From the broth 3.7 g pravastatin dibenzylamine salt crude product was produced by the method given in Example 4. from which after recrystallization 2.9 g pravastatin dibenzylamine salt was obtained. The recrystallized pravastatin dibenzylamine salt was suspended in 45 ml ethanol, then under stirring 110 mole% sodium hydroxide was added by the feeding of 1M ethanolic sodium hydroxide solution. Stirring of the solution is continued for half an hour, then 0.3 g charcoal was added into it and stirred for another half an hour. The solution was filtered, and the filtrate was concentrated to 15 ml. Then 60 ml acetone was added to the concentrate at 56-60°C. The solution obtained was cooled to room temperature, then kept overnight at +5°C. Subsequently, the precipitate was filtered, then washed with 2x20 ml acetone, 2x20 mi cthyl acetate and 2x20 mi n-hexane, and fmally dried mn vacum. The resulting 1.7 g crude pravastatin was dissolved in ethanol, then clarified with charcoal and crystallized from an ethamol-cthyl acetate mixtore. In this way 1.54 g pravastatin was obtained that was identical with the product of Example 2.

Example 6 oo : As desc: Jbed in Example 4, from the slant culture of ** = Aortierella maculata n. spec.

E-97 [NCAIM(P)F 001266] strain cultivated for 7-10 days, a 500 mi inoculum medium MI sterilized in a 3000 ml Erlenmeyer flask was inoculated and incubated at 28°C for 3 days on a rotary shaker. ! Composition of the medium MI: oo oT glucose 40g casein 5g

KCl 0.5g

NaNO; 3g

KH,PO, 2g

MgSO, x 7H,0 0.5g

FeSO, x 7H.O 0.0ig in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 6.0 and the sterilization is ) carried out at 121°C for 35 min. The seed culture obtained is inoculated into 5 liters bioconversion medium P12 sterilized in a fermenter.

Composition of the medium P12: glucose 10g malt extract 50g yeast extract 5g corn steep liquor Sg

MgSO, x 7H.O lg

Tween-80 0.5g - in 1000 ml tap water.

Before the sterilization the pH of the medium is adjusted to 7.0, then the sterilization was carried out at 121°C for 45 min. The fermemation. substrate feeding and bioconversion were _ carried out according to the Example 2. After finishing the bioconversion the pravastatin formed in the concentration of 620 ng/ml was isolated as follows: )

The pH of 5.15 liters broth containing 62( xg/ml pravastatin was adjusted with 2M sodium hydroxide to 9.5 value then stirred at room temperature for 1 hour. The broth was filtered and the mycelium was washed with suspension in 1x2 liters and then 1x0.5 liters water. Filtrates are combined and the pH of the aqueous solution was adjusted with 20% sulfuric acid to 3.7 value and . extracted with 2.5 liters then with 1.5 liters ethyl acetate. The ethyl acetate extracts were combined, washed with 2x0.5 liters water and 1.95 g dicyclohexylamine was added. The ethyl acetate extract was concentrated at 40°C to 200 ml under reduced pressure, and 0.195 g dicyclohexylamine was added again into the concentrate, which was then stirred at 15°C for 6 hours. The precipitated crystalline material was filtered, washed with 20 ml and with 15 mi ethyl acetate and dried at 40°C.

In this way 3.51 g crude product was obtained. After the recrystallization of the crude product in an acetone - ethanol mixture, 3.05 g of pravastatin dicyclohexylamine salt was obtained (melting point: 162-168 °C), which was converted to pravastatin according to the Example 5.

Example 7

The fermentation, substrate feeding and bioconversion were carried out with the

Mortierella maculata n. spec. E-97 [NCAIM(P)F 001266] strain as described in Example 2.

Pravastatin obtained as a result of the bioconversion is isolated from the broth as follows. $ liters culture broth containing in concentration 650 ng/ml pravastatin was filtered on a filter cloth. The mycelium of the mold was stirred in 2 liters 0.1M sodium hydroxide solution for an hour. then filtered. The two filtrates were combined and the pH was adjusted with 15% sulfuric acid to 3.5-4.0. Subsequently, the solution was extracted with 2x1.8 liters ethyl acetate. The combined ethyl acetate phases were washed with 800 ml water. Then 400 mi deionized water was added and the pH of the mixture is adjusted by 1M sodium hydroxide 10 a 8.0-8.5 value. The mixture was stirred for 15 minutes, then the phases were separated. 300 ml water was added to the ethyl acetate phase and the pH are adjusted to 8.0-8.5. After stirring for 15 minutes the phases were separated. 300 mi water was added again 0 the ethyl acetate phase and the pH was adjusted 10 8.0- 9.5. Then the mixture was stirred for 15 man. The two phases were separated again. All aqueous phases were combined and the pH arc adjusted with 15% sulfuric acid 10 3.54.0, then extracted with 3x300 ml ethyl acetate. The combined ethyl acetate extracts were washed with 150 mi water, dried with anhydrous sodium sulfate, and filtered. Then 150 mole% dioctylamine—calculated for the pravastatin content as added to the ethyl acetate extract. The ethyl acetate was evaporated to about 1/10 volume and acetone vias added until precipitation. The mixture was kept at'&5°C overnight.

The precipitate was filtered on a G-4 filter, washed with 20 ml acetone and then with 20 ml n-hexane and dried in vacuum at room temperature. The 3.3 g crude pravastatin dioctylamine salt obtained was recrystallized from 20 ml acetone resulting in 2.7 g pure pravastatin dioctylamine salt. Melting point: =#31 143-146°C. The pravastatin dioctylamine salt was converted {6 pravastatin with the method given in

Example 5.

Example 8

By the development of the hydroxylation ability of Mortierella maculata n. spec. E-97 strain isolated from natural habitat, which is able to 6B-hydroxylate compactin. in the mutation- selection and enzyme induction experiments discussed in detail below, Mortierella maculata n. sp. E- 97/15/13 [NCAIM(P)F 001267] mutant strain was produced. .

Mortierella maculata n. sp. E-97 [NCAIM(P)F 001266] strain isolated by us was cultivated on MS slant agar medium at 28°C for 7 days.

Composition of agar medium MS: glucose 4g malt extract 10g yeast extract 4g agar 20g in 1000 ml distilled water.

Spores were washed off from the slant cultures by 5 ml 0.9% sodium chloride solution. then after transferring the spore suspension into a sterile Petri dish it was irradiated by ultraviolet light for 1 minute. Subsequently, N-methyl-N'-nitro-N-nitrosoguanidine was added to the spore suspension in the final concentration of 2000 «g/ml. Then the suspension was transferred into a 100 ml Erlenmeyer flask and it was shaken at 28°C with a rate of 150 rpm for 20 min.

Subsequently, the spores were sedimented by centrifugation with a rate of 4000 rpm for 10 min, then suspended in sterile 0.9% sodium chloride solution. The suspension was spread on an agar plate MU-

VB containing 10 ug/ml benomy! and 1% defibrillated blood.

Composition of agar medium MU-VB: glucose 40g asparagine 2g peptone 2.5g potassium dihydrogen phosphate 0.5g agar 20g in 990 ml distilled water; after sterilization the medium was completed with 10 ml bovine biood and mg benomyl.

The agar plates were incubated at 28°C for 7 days, then the grown colonies were transferred by random selection into test tubes containing agar medium PS.

Composition of agar medium PS: glucose 40g mycological peptone 10g agar 15g in 1000 mi distilled water.

Before sterilization the pH of the medium is adjusted to 5.6-5.7 value. The sterilization is carried out at 121°C for 20 min.

Slant cultures were incubated at 28 °C for 12 days, and their pravastatin productivity was tested in shaken flask experiments as described in Example |. Mortierella maculata n. sp. E- 97/15/13 mutant strain was sclected by this method. which yielded pravastatin exceeding 60% conversion rate from the applied compactin acid sodium salt substrate being in the concentration of 1000 ug/ml.

The hydroxylase enzyme of Mortiereila macslata n. sp. E-97/15/13 stxain was induced by the cultivation on MU-VB agar medium containing 100 ug/ml 8-de-(2-methyl-butyryl) compactin and/or compactin. After random selection of the grown colonies they were transferred into inducer containing MU-VB slants. Pravastatin productivity of the grown slant cultures was examined by the method written in the Example | with the difference that the compactin substrate feeding in the quantity of 500 ng/ml was carried out from the 4th day of the fermentation for further 11 days and the compactin sodium substrate was added gradually during the twelve days converted completely to pravastatin.

By the end of the bioconversion carried out in 50 shake flask cultures from 30 g comg..tin sodium substrate the formation of 18.5 g pravastatin was measured by HPLC.

Recovery of fe pravastatin from the combined fermentation broths was carried 68 according to the following method.

: After finishing the fermentation the pH of 5.5 liters broth with a pravastatin concentration of 3360 ng/ml was adjusted with 20% sulfuric acid solution to 3.5-3.7. Subsequently,

the acidic solution was extracted by 2.75 liters ethyl acetate.

Phases were separated, and a clear extract was prepared by centrifugation from the emulsified organic phase.

Broth was extracted two more times with 1.37 liters ethyl acetate as previously described.

The combined ethyl acetate extracts were washed with 2 x 1.15 liters deionized water. then 150 mole% dibenzylamine-- calculated for the .

pravastatin content measured by HPLC--was added to the ethyl acetate solution.

The ethyl acetate solution was concentrated in vacuum to about 0.23 liters volume.

Further 20 mole% dibenzylamine was added to the concentrate and the precipitate solution was kept overnight at 0-5°C.

Precipitated pravastatin acid dibenzylamine salt was filtered. then the precipitate was washed by suspending it in cooled ethyl acetate and ther: two times in n-hexane. finally dried at 40-50°C in vacuum.

The crude product obtained (22.4 g) was dissolved in 0.67 liters acetone at 62-66°C temperature, and the solution was clarified with 2.2 g charcoal.

After the clarification the acetone filtrate was concentrated in v.cuum to 0.56 liters volume.

Crystals precipitated from the concentrate were dissolved again at }

the above temperature, then the solution was cooled 10 room temperature.

Subsequently, the recrystallization was continued overnight at 0-5°C.

Precipitated crystals were filtered, and washed by suspension two times in cooled acetone and two times in n-hexane.

Recrystallized pravastatin acid © dibenzylamine salt was dried :n vacuum at 40-50°C.

Pravastatin acid dibenzylamine salt obtained

(14.8 g) was dissolved at 40-44°C in 740 ml ethyl acetate-ethanol (9:1) mixture, then 110 mole%

sodium hydroxide was added to the solution in form of a IM ethanolic solution.

Stirring of the obtained precipitated solution was continued for half an hour at room temperature, then a complete precipitation was achieved as a result of the application or ice cooling for 1-1.5 hours. Subsequently, the precipitate was filtered and washed with 2 x 150 ml cooled ethyl acetate and 2x150 ml n-hexane, } finally dried in vacuum at 40-50°C. The pravastatin obtained was dissolved in ethanol, clarified by 1.0 g charcoal, then crystallized from ethanol-ethyl acetate mixture. This way 9.4 g pravastatin was obtained, with physical constants corresponding to the data given in Example 2.

Although certain presently preferred embodiments of the invention have been described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the described embodiments may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law.

Claims (37)

CLAIMS Wwe claim: a

1. A microbial process for the preparat.on of the compound of formula (I) " = a WOH . - . ‘ NaluC BY oo "HO, _} 8 J WN f T H CH; ~ CH3 (J HO < A MD from a substrate compound of formula (11), JOH ROOC - - HO 0 ’ ‘7, H eo TH CH; CHj; (In wherein R stands for an alkali metal or ammonium ion. comprising the steps of a) cultivating a strain of Mortierella maculata filamentous mold species ak: _ to 6- hydroxylate a compound of formula (II) on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts, b) feeding the substrate to be transformed into the developed culture of Mortierella maculata, c) fermenting the substrate until the end of bioconversion, d) separating the compound of formula (I) from the culture broth, and e) isolating the compound of formula (I).

2. The process of claim 1, wherein said medium is a nutrient broth.

3. The process of claim 2, wherein said step of separating the compound of formula (I) from the culture broth is performed by adsorption on an anionic ion exchange resin.

4, The process of claim 2, wherein said step of separating the compound of formula (I) from the culture broth is performed by extraction with a water immiscible organic solvent, followed by the preparation of its lactone derivative or its secondary amine salt as an intermediate.

S. The process of claim 2. wherein said step of separating the compound of formula (I) from the culture broth is performed by purification of the alkaline aqueous extract obtained from the organic solvent extract of the fermentation broth with chromatography on a non-ionic adsorbing resin.

6. The process of claim 1, wherein the strain of Mortierella maculata is the Mortierella maculata n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001266.

7 The process of claim 1, wherein the strain of Mortierella maculata is the Mortierella maculata n. sp. E-97/15/13 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession mumber NCAIM(P)F 001267.

8. The process of claim 1, wherein the hydroxylase enzyme of the strain used for the transformation is induced by 8-de(2-methyl-butyryl) corpactin or compactin.

9. The process of claim 2, wherein a compound of formula (II) as a substrate is added into the culture parallel with the feeding step, and wherein the feeding step depends on the pH of the

_. culture and itsquantitysis 0.5-1.0% related to the volume of the broth. C- PT 28 . : SEE

10. The process of claim 1. wherein the fermentation step is carried out on a medium containing a carbon source selected from the group consisting of glucose. fructose and glycerine.

11. The process of claim 1. wherein the fermentation step is carried out on a medium containing a nitrogen source selected from the group of consisting of soybean meal. peptone. casein, yeast extract and meat extract.

12. The process of claim 2. wherein the compound of formula (I) formed during the bioconversion is separated from the culture broth by adsorption from the filtrate of the broth and from the washing water 6f the mycelium on an anion exchange resin. eluting the compound of formula (I) from the resin. transforming the compound of formula (I) completely to its lactone form. isolating the lactone derivative, hydrolyzing the lactone derivative by sodium hydroxide. and desalting the compound of formula (I) on a non-ionic adsorption resin.

13. The process of claim 12, wherein said anion exchange resin has quaternary ammonium . active groups carrying polystyrene-divinyibenzene skeleton. is used for the separation of the compound of formula (I) from the filtrate of the broth.

14. The process of claim 4, wherein the compound of formula (I) formed during the bioconversion is extracted in acid form from the broth, the broth having been acidified to the pH of

3.5-3.7, or from filtrate of the broth by a water-immiscible organic solvent.

AN ’ Co WO 00/4617 PCT/US00/02993

15. The process of claim 14 wherein said water-immiscible organic solvent is ethyl acetate.

16. The process of claim 14 wherein said water immiscible organic solvent is isobutyl acetate.

17. The process of claim 5, wherein said compound of formula (I) is extracted in sodium salt form from the organic solvent by aqueous sodium hydroxide solution, and purified on a non-ionic adsorption resin.

18. The process of claim 14, wherein said compound of formula (I) is precipitated from the extract in crystalline form with a secondary amine containing alkyl-, cycloalkyl-, aralkyl- or aryl- substituents.

19. The process of claim 18. wherein the crystalline secondary amine salt is suspended in a mixture of a 1-4 carbon atom-containing alkyl ester of acetic acid and water; an equivalent quantity of sodium hydroxide is added in aqueous solution to the suspension such that an organic phase and an aqueous phase are formed; the organic and aqueous phases are separated; the aqueous phase is washed with isobutyl acetate. then clarified with activated carbon: and the aqueous solution is lyophilized.

20. The process of claim 19, wherein said alkyl ester is isobutyl ester.

21. The process of claim 18, wherein the crystalline secondary amine salt is suspended in a 1-4 carbon atom-containing alcohol; from the suspension a solution of the compound of formula (I) is prepared by adding an ethanolic solution of sodiam hydroxide; and the compound of formula (I) is precipitated from the solution by acetone.

22. The process of claim 21, wherein said 1-4 carbon atum-containing alcohol is ethanol.

23. The process of claim 18. wherein the crystalline secondary amine salt is dissolved in a mixture of a 1-4 carbon atom-containing alkyl ester of a 1-4 carbon atom-containing alkane carboxylic acid and a 1-4 carbon atom-containing alcohol; and fror= the solution the compound of formula (I) i precipitated in crystalline form by adding sodium hydroxide. )

24. The process of claim 23, wherein said mixture is an ethyl acetate-ethanol mixture.

25. The process of claim 19. wherein pravastatin is isolated from the fermentation broth via the dibenzylamine salt of the acid form of the compound of formula (1).

26. The process of claim 19. wherein the acid derivative of the compound of formula (I) 1s purified through its dicyclohexylamine salt.

27. The process of claim 19. wherein the acid derivative of the compound of formula (I) is purified through its dioctylamine salt.

28. The process of claim 19. wherein the compound of formula (I) is purified t: at least

99.5%, as measured by HPLC. using gel chromatography.

29. The process of claim 1. wherein said strain of Mortierella maculata is cultivated at about 25° to about 30°C.

30. A biologically pure culture of the Mortierella maculata n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001266.

. ‘. : 31. A biologically pure culture of the Mortierella maculata n. sp. E-97/15/13 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, i Hungary under accession number NCAIM(P)F 001267.

32. A Mortierella culture able to 6p-hydroxylate a compound of formula (II) JOH ROOC ” HO 0] “In Ye T H CH; _~ CH; an wherein R stands for an alkali metal or ammonium ion. consisting essentially of a novel strain Mortierella maculata n. sp. E-97 deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest. Hungary under accession number NCAIM(P)F 001266.

33. A Mortierella culnre able to 6f-hydroxylate a compound of formula (11)

. WOH rooc” NV" © = HO Oo “ny eo T H i J CH; A CHs 0 : Zw 0 - . aK H . =“ (IN wherein R stands for an alkali metal or ammonium ion. consisting essentially of a novel strain ) Mortierella maculata n. sp.

E-97/15/13 deposited at the National Collection of Agricultural and Industrial Microorganisms.

Budapest.

Hungary under accession number NCAIM(P)F 001267.

34. The sodium salt of pravastatin in a crystalline form.

35. The sodium salt of pravastatin according to claim 34, wherein the melting point is in the range of from about 170°C to about 173°C.

36. Process for the preparation of sodium salt of pravastatin in a crystalline form, comprising the steps of: 2) providing a solution containing pravastatin and sodium cations in a lower aliphatic alcohol; oo ~b) adding ethyl acetate to said solution; oo c) crystallizing sodium salt of pravastatin from said solution.

37. The process according to claim 36 wherein said lower aliphatic alcohol is ethanol. oo © Amended Sheet 30 July 2002