HU213349B - Method for the preparation of macrolide compounds by fermentation - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention relates to compounds of formula (I) and their salts, wherein R 1 is methyl, hydroxymethyl or carboxyl, R 1 is C 1-4 alkyl, R 2 is hydroxy when R 3 is hydrogen or R 2. and R3 together with the carbon atom to which is attached> C = O or> C = NOR9 General R7 HU 213 349 BA Description: 9 pages (including 1 sheet)

Description

Scope of the description: 9 pages (including 1 page figure)

EN 213 349 B

By treating a compound of formula (2), wherein R ¹ , R ² , R ³ and R ⁴ are as defined herein, with a steptomyces or Absidia genus capable of selectively oxidizing a compound of formula (2); or, in the presence of a composition derived from a microorganism containing the enzyme, and, if desired, to prepare compounds of formula (I) wherein X is C = NOR ^{9, a} corresponding 23-oxo compound of formula (1) H ₂ NOR ⁹ , where R ⁹ is as defined above, reacts and, if desired, converts the resulting compound of formula (1) into a salt.

The present invention relates to a process for the preparation of novel intermediate macrolide compounds by fermentation.

British Patent Specification 2 166 436 and European Patent Application 0170 006 disclose a group of macrolides, designated S541 and LL-F28249. Derivatives of these compounds are described, for example, in British Patent Publication Nos. 2,176,182 and 2,192,630.

Pesticide compounds having a structurally close relationship to the above compounds are disclosed in Hungarian Patent No. 206,720. The compounds of the present invention are primarily useful in the preparation of such macrolide compounds.

The present invention therefore relates to a process for the preparation of compounds of formula (I) and salts thereof, wherein R is methyl, hydroxymethyl or carboxyl;

R ¹ is 1 = C 1 alkyl;

R ² is hydroxy when R ³ is hydrogen or

R ² and R ³ together with the carbon to which they are attached represent a> C = O or> C = ^NOR9 group - wherein ^R9 is C1-4 alkilcspoport, or> C = NOR ⁹ residue is E-configuration it is

R ⁴ is hydroxy or C 1-4 alkoxy,

R ⁶ is hydrogen or hydroxy, with the proviso that when R ⁶ is hydrogen then R is hydroxymethyl or carboxyl, and when R ⁶ is hydroxy, R is methyl,

^R7 is hydrogen or hydroxy, such that a compound of formula (2)

- wherein R ¹ , R ² , R ³ and R ⁴ are as defined in the specification in the presence of a microorganism of the genus Streptomyces or Absidia capable of selectively oxidizing the compound of formula (2), or an enzyme derived therefrom, or if desired, to prepare compounds of formula (I) wherein X is> C = NOR ⁹ , a corresponding 23-oxo compound of formula (1) is reacted with a compound of formula H ₂ NOR ⁹ , wherein R ^{9 is as} defined above. and, if desired, converting the resulting compound of formula (1) into a salt.

Examples of C 1 -C 4 alkyl in R ¹ or R ⁹ include methyl, ethyl, η-propyl, isopropyl, η-butyl, isobutyl or tert-butyl.

Compounds of formula (I) containing an acidic group may form salts with suitable bases, such as alkali metal salts such as sodium and potassium salts.

In the compounds of formula (1), R ^{1 is} preferably isopropyl.

An important subgroup of compounds of formula (1) are those wherein R ² is hydroxy and R ^{3 is} hydrogen; or R ² and R ³ together with the carbon atom to which they are attached> C = O.

From the point of view of efficacy, the compounds of the invention where:

R is carboxy, R ¹ is isopropyl; R ² and R ³ together represent> C = NOCH ₃ and R ⁶ and R ⁷ are hydrogen.

Compounds of formula (I) in which R is carboxyl are particularly useful as intermediates in the preparation of other macrolide compounds.

The compounds of the invention may be prepared by a number of methods; these are described below, wherein R, R 1, R 2, R 3, R 4, R 6 and R 7 are as defined above for formula (1) unless a specific comment is made in this respect. In some processes, it may be necessary to protect one or more hydroxyl groups present in the starting material before carrying out the reaction. In these cases, after carrying out the reaction, it may be necessary to remove the protecting group of the same hydroxyl group (s) to produce the compound of the invention. General methods for introducing and removing a protecting group can be found, for example, in T.W. Green: "Protective Groups in Organic Synthesis" Wiley-Interscience, New York 1981; and J.F.W. McOmie: "Protective Groups in Organic Chemistry, Plenum Press, London 1973. The acetyl group can be removed, for example, by basic hydrolysis such as sodium hydroxide, potassium hydroxide or ammonia in an aqueous alcohol such as methanol.

The compounds of formula (1) according to the invention are prepared by reacting a compound of formula (2) with a microorganism belonging to the genus Streptomyces or Absidia, or an enzyme derived therefrom, from the 4 and / or 12 positions of the molecule. incubation in the presence of a composition containing the same.

The microorganisms and extracts thereof suitable for use in the process of the present invention can be identified by preliminary, small-scale studies, and can be ascertained.

EN 213 349 to convert compounds of formula (2) into compounds of formula (1). The formation of compounds of formula (1) can be confirmed by appropriate chromatographic analysis of the reaction mixture (e.g., high performance liquid chromatography (HPCL)).

It has been found that microorganisms belonging to the genus Streptomyces and Absidia and extracts thereof are particularly suitable for use in the method of the invention.

For example, the following strains of Streptomyces griseoplanus, Streptomyces Virgini, Streptomyces cacaoi, Streptomyces spinichromogenes var. pimycycetes, Streptomyces tendae, Streptomyces aureofaciens, Streptomyces autotrophicus, Streptomyces filamentosus, Streptomyces canescens, Streptomyces deltas, Streptomyces füngicidicus var. espinomycetcicus, Streptomyces mycarofaciens, Streptomyces rimosus, Streptomyces djakertensis, Streptomyces mashuensis and Streptomyces platensis subsp. malvinus and mutants of these strains.

The following strains and mutants of Streptomyces mashuensis, Streptomyces rimosus and Streptomyces platensis subsp. for example, Malvinus, for example, Streptomyces mashuensis ISP 5221, Streptomyces rimosus NRRL 2455 and Streptomyces platensis subsp. malvinus NRRL 3761.

The mutants of the above strains may be generated spontaneously or may be produced by a number of methods described in British Patent No. 2,166,436.

Biotransformation (bioconversion) may also be carried out with a microorganism comprising the genetic material of one of the above-mentioned microorganisms involved in the synthesis of the compounds of formula (1). Such microorganisms can be produced by genetic engineering (gene manipulation); for a description of these, see D. A. Hopwood: "Cloning Genes Forum Antibiotic Biosynthesis in Streptomyces Spp. Production of a Hybrid Antibiotic, Microbiology, L. L. Lieve, American Society of Microbiolgy, Washington, O.C., pp. 409-403. 1985. These methods can be applied in the same way as the methods previously described for cloning biosynthetic genes with antibiotic activity; such biosynthetic genes were used, for example, actinorodine% F. Malpartida and D. A. Hopwood, Natúre 309, 462 (1984), erythromycin [R. Stanzák et al., Biotechnology 4,229 (1986)] and an important enzyme in the production of penicillin and cephalosporin [S. M. Sansom et al., Natúré, 318, 191 (1985)].

The enzymes useful in the process of the invention can be obtained from a wide variety of sources; however, the above-mentioned Streptomyces microorganisms are a particularly useful source for converting compounds of formula (2) into compounds of formula (1).

According to a preferred embodiment of process A according to the invention, a compound of formula (2) is converted to a compound of formula (1) by, for example, feeding the compound of formula (2) in a solvent in a fermentation medium containing the above-mentioned microorganism. and in the presence of nitrogen spinning and mineral salts. As a source of assimilable carbon and nitrogen, and as a source of minerals, simple or compound nutrients can be used. As a carbon source, for example, glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils are commonly used. The carbon source is generally used in an amount of 0.5-10% by weight of the fermentation medium.

As a nitrogen source, soy bean flour, corn, distillation soluble substances, yeast extract, cottonseed flour, peptones, ground peanut flour, malt extract, molasses, casein, amino acid mixture, ammonia (in gas or solution), ammonium salts and nitrates are commonly used; Urea or other amides may also be used. The nitrogen source is generally used in an amount of 0.1 to 10% by weight of the fermentation medium.

Mineral salts, which can be embedded in the culture medium, are generally salts which are sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, and the like. , copper, molybdenum, boron, phosphate, sulfate, chloride and carbonate ion requirements.

An anti-foaming agent, which is added to the medium at the desired intervals, can be used to prevent foaming of the fermentation medium.

During the addition, the compound of formula (2) can be used to dissolve a water-miscible organic solvent (e.g. an alcohol such as methanol or isopropanol, a diode such as 1,2-propanediol or 1,3-butanediol; acetone; nitrile (e.g. acetonitrile); ether (e.g., tetrahydrofuran or dioxane); substituted acid amide (e.g. dimethylformamide) or dialkyl sulfoxide (e.g. dimethylsulfoxide). at the beginning of cultivation or in general when the microorganism is already being propagated, for example 2-4 days after the start of the culture.

The cultivation of the microorganism is generally carried out at a temperature of from 20 to 50 ° C, preferably from 25 to 40 ° C, if desired by aeration and stirring, for example by shaking or stirring. Alternatively, the medium may be inoculated with a small amount of suspension of the spore-forming microorganism; however, in order to avoid delays, a vegetative graft can be produced from the microorganism by inoculating a small amount of the culture medium with the spore form of the microorganism and transferring the resulting vegetative graft into the fermentation medium; or, more preferably, one or more grafts are allowed to proliferate before being transferred to the fermentation medium. The fermentation is generally carried out at pH 4.0-9.5, preferably 5.5-8.5 when using the Streptomyces strain; and preferably

EN 213 349

We work at pH 4.0 to 8.0 when using other bacteria or fungi.

After addition of the compound of formula (2) to the culture, which is usually done by gentle agitation, the culture is continued to increase the amount of desired product. The presence of the product in the fermentation broth can be determined by examining the fermentation broth extract by HPLC and UV spectroscopy at 238 nm.

The product (s) from the whole fermentation broth can be carried out by conventional isolation and separation techniques as described in U.S. Patent Nos. 2,166,436 and 2,176,182.

If plant cells are used in the fermentation process, the culture is preferably carried out by culturing the plant cell growth regulator such as indole acetic acid, naphthyl acetic acid or indole butyric acid, 2,4-dichlorophenoxyacetic acid, kinetin or benzyl. working in an aminopurine-containing medium at 15-35 ° C and maintaining the pH between 5.0 and 7.5. Ammonium salts and nitrates are thus a preferred source of nitrogen in the fermentation medium. Sucrose, phosphorose and glucose are preferred carbon sources in the fermentation medium.

In another preferred embodiment of process A of the present invention, a compound of formula (2) can be converted to a compound of formula (I) by combining a compound of formula (2), for example, in a suitable solvent and incubating with an enzyme composition of the invention, optionally in a buffer solution. , for example, 0-60 ° C, preferably 20 ° C to 40 ° C, for example about 28 ° C. This reaction is generally carried out at a pH range of 3.5 to 8.5, for example 5.5 to 7.5. If desired, the reaction may be carried out in the presence of a cofactor such as NADH or NADPH. When the reaction is complete, if the conversion of the compound of formula (2) to the compound of the invention is stopped, this is determined by examining the extracts from the reaction mixture by HPLC and UV spectroscopy at 238 nm - then the product is isolated by standard isolation and purification. obtained by isolation methods described in British Patent Nos. 2,166,436 and 2,176,182.

For example, the enzyme useful in the method of the invention may be prepared from a culture of a microorganism which produces the enzyme in a medium. Suitable media and fermentation conditions used for the preparation of the enzyme have been described above for the preparation of compounds of formula (1) from compounds of formula (2) in the presence of a microorganism. The time at which the desired enzyme activity reaches its maximum will, of course, vary according to the microorganism used, and it is therefore desirable to determine the optimum time for cultivation independently of each strain used.

In the case of microorganisms where the enzyme is extracellular, the liquid culture medium or the filtrate obtained after removal of the entire cell may be used as the source of the enzyme. If the enzyme is cell-bound, it can be released by conventional methods, for example by means of sound vibrations, grinding with glass beads, homogenization, lytic enzymes or detergents, after suspending the cells in a suitable buffer solution. The composition thus obtained can be used as an enzyme source, either together with the cell debris or after its removal. Preferably, however, the enzyme is further purified by conventional methods. For this purpose, batch or column chromatography, ion exchange celluloses or affinity adsorbents, or other adsorbents, such as hydroxyl apatite, can be used. In addition, the enzyme may be concentrated or further purified by molecular sieves or ultrafiltration or by salting. In the purification process, it is generally desirable to keep the pH between 3 and 11.

The enzyme can also be used in immobilized form: for example, it can be rendered insoluble by the use of an appropriate matrix, or linked to the matrix, for example, by coupling the enzyme extract to an otherwise inert, inorganic or organic polymer, or by incorporating it into a fiber or a membrane or polymer, for example, bonded to a polyacrylamide gel: adsorbed on an ion exchange resin crosslinked with a suitable reagent such as glutaraldehyde; or covered with a sheath, for example, into a bead shape. The immobilized enzymes are advantageously used in both batch processes, after which the enzyme is again used in continuous processes, whereby the substrates pass through the column containing the immobilized enzyme.

According to the process of the present invention, compounds of formula (I) wherein: X is> C = NOR ⁹ can be prepared by reacting a corresponding 23-oxo derivative of formula (I) with a compound of formula H ₂ NOR ^9. wherein R ⁹ is as defined above.

This oxime-forming reaction may conveniently be carried out at a temperature of from -20 ° C to 100 ° C, for example from -10 ° C to 50 ° C. The reagent H ₂ NOR ⁹ is preferably used in the form of a salt such as an acid addition salt such as its hydrochloride. When such a salt is used for this reaction, it is preferred to work in the presence of an acid scavenger.

Examples of solvents for this reaction include: alcohols such as methanol and ethanol: amides such as dimethylformamide, dimethylacetamide and hexamethylphosphoric acid triamide; ethers such as cyclic ethers such as tetrahydrofuran or dioxane, or linear ethers such as dimethoxyethane or diethyl ether; nitriles such as acetonitrile; sulfones such as sulfolane; hydrocarbons such as halogenated hydrocarbons such as dichloromethane; and a mixture of two or more of the above solvents. Water may also be used as a co-solvent.

If water is used as the solvent for the reaction, the reaction mixture can be conveniently buffered with an appropriate acid, base or buffer solution. Suitable acids for this purpose include, for example, mineral acids such as hydrochloric acid and sulfuric acid; and carboxylic acids such as acetic acid. Bázis4

For example, alkali metal carbonates and hydrogen carbonates such as sodium bicarbonate may be used; hydroxides such as sodium hydroxide; and alkali metal salts of carboxylic acids, such as sodium acetate. Preferably, a mixture of acetic acid and sodium acetate can be used as a buffer.

The salts of the acids of formula (1) can be prepared by the usual method, for example by treating the acid with a base or by subtracting one of the salts of the acid into an other salt by ion exchange.

Intermediates of formula (2) in which: R ² is hydroxy and then R ^{3 is} hydrogen; or R ² and R ³ together with the carbon atom to which they are attached form C = O, known compounds disclosed in British Patent Specifications 2 166 436 and 2 176 182 and European Patent Specification 0 215 654 or from known compounds by processes described above.

Intermediates of formula (2) in which R ² and R ³ together are > C = NOR ⁹ are known from compounds of formula (2) as described above in U.S. Patent Nos. 2,166,336 and 2,176,182. can be prepared by the methods described in U.S. Patent Nos. 2,192,630 and 0,231,104.

The present invention is described in detail in the following non-limiting embodiments.

In these examples, the compound of formula (2) wherein R ^{1 is} isopropyl, R ^{2 is} hydroxy and R ^{3 is} hydrogen, R ^{4 is} hydroxyl, "Factor A". The compounds of the present invention are named with respect to factor A. In the examples, all temperatures are given in degrees Celsius, mass spectra were recorded using an electron beam method.

Example 1

Preparation of 12-desmethyl- (Factor A) -12-carboxylic acid Streptomyces platensis subsp. Malvinus NRRL 3761 (ATCC 29778) was treated with 5 ml of sterile water and 25 ml of "A medium" in 250 ml volumetric flasks was added to the resulting mixture. The composition of medium A is as follows:

g / L D-glucose 2.5 Malt dextrose MD30E 25.0 Arka soy 50 12.5 Molasses 1.5 Potassium dihydrogen phosphate 0,125 Calcium carbonate 1.25 3- (N-morpholinyl) propanesulfonic acid 21.0 Distilled water as is required

Before autoclaving, the pH was adjusted to 6.5 with sulfuric acid.

The flask was incubated at 250 rpm on a 250 rpm shaker and 100 ml of the 2 day culture was used to inoculate 5 liters of a 7 liter fermenter containing medium A. Incubation is continued at 2 ° C / min with stirring at 250 rpm, and at 28 ° C, and after 2 days, a solution of 2.5 g of factor A in 50 ml of dimethyl sulfoxide is added. The fermentation was continued for a further 5 days, after which the cells were removed by centrifugation and extracted with methanol. After removal of the cells, the aqueous supernatant was extracted with ethyl acetate, the ethyl acetate extracts were combined, added to the methanol extract, and evaporated. The resulting oil-like product was dissolved in water and the 160 mL solution was washed with ether and extracted with ethyl acetate. The combined ethyl acetate extract was evaporated, the residue was dissolved in 15 ml of acetonitrile, centrifuged, and then applied to a Spherisorb S5 ODS-2 column (250 mm x 20 mm). Detection was performed at 240 nm in 1 ml portions diluted with a mixture of equal volumes of acetonitrile and 0.1M ammonium dihydrogen phosphate solution 1: 1. A mixture of acetonitrile and 0.1 M ammonium dihydrogen phosphate solution (1: 1) at a constant flow rate of 25 ml / min was used as eluant and the eluate of the peak between 14 and 17 minutes was collected. All such fractions were combined, diluted with equal volume of water and pumped back to the top of the column. The column was eluted with acetonitrile, the acetonitrile removed in vacuo, the solid residue dissolved in acetone, diluted with cyclohexane, and lyophilized. 148 mg of the title compound are obtained as a colorless solid.

1 H-NMR (CDCl ₃ , δppm, 200 MHz): 0.79 (d7.3H), 0.94 (d7.3H), 1.04 (d7.3H), 1.52 (s, 3H ), 1.59 (s, 3H), 1.84 (s, 3H), 3.23 (m, 2H), 3.73 (dl 1.1H), 3.92 (d6, 1H), 4, 27 (d6, 1H), 5.08 (t8, 1H) and 5.18 (d9, 1H);

IR (CH ₃ Br) 1702, 1730 and 3480 cm ^-1 ;

The mass pattern shows M ⁺ ion at m / z 642; as well as fragment ions at m / z: 624,606, 560, 514, 512, 496, 478, 455, 437, 384, 344, 297, 278, 265,247, 237,219, 181 and 95.

¹³ C NMR (octm 25.0 MHz): 10.8 (q), 13.7 (q), 14.8 (q), 19.6 (q), 22.6 (q), 26.6 (d), 34.6 (t), 35.7 (d), 40.5 (t), 42.5 (t), 45.3 (d), 46.6 (d), 67 , 3 (d), 67.6 (d), 68.0 (t), 69.12 (d), 76.5 (d), 79.1 (d), 80.0 (s), 99, 5 (s), 117.6 (d), 119.3 (d), 122.4 (d), 127.3 (d), 130.2 (s), 132.5 (d), 134.2 (s), 137.2 (s), 137.4 (s), 141.7 (s), 173.0 (s) and 177.1 (s).

Example 2

Preparation of 12-desmethyl-23-oxo (Factor A) -12-carboxylic acid 721 mg of 23-oxo (factor A) compound (prepared according to example 21 of British Patent No. 2,176,182) are prepared in 50 ml of methanol. dissolved in the culture as described in Example 1, fermentation was carried out under the same conditions for 2 days, and the cells were removed by centrifugation. The pH of the supernatant was adjusted to 2.0 with concentrated hydrochloric acid and extracted with ethyl acetate. The ethyl acetate extract was evaporated and the oily residue was taken up in 100 ml of water containing 2 g of sodium bicarbonate.

21 213 349 9. The solution was washed with ether, adjusted to pH 2.0 and extracted with ether. After aggregation, the ether extract was evaporated to give a semi-solid which was dissolved in methanol (4 mL) and treated with 0.7 mL of acetonitrile and 0.1 M ammonium dihydrogen phosphate solution (1: 1) and filtered.

The resulting solution was applied to a Spherisorb S5 ODS-2 column of 250 mm x 20 mm in 1.775 ml portions and the fractions were detected at 255 nm. As a eluent, a 1: 1 mixture of acetonitrile and 0.1 M ammonium dihydrogen phosphate solution was used at a constant flow rate of 25 mL / min; Fractions containing detected peaks from different injections between 12.2 and 14.9 minutes were collected, diluted with equal volume of water and pumped back to the top of the column. The column was washed with a 1: 3 mixture of acetonitrile and water and eluted with acetonitrile.

The acetonitrile solution was evaporated, acetone and cyclohexane were added to the residue and lyophilized. 25 mg of the title compound are obtained as a colorless solid.

1 H-NMR (CDCl ₃ , ppm, 200 MHz): 0.86 (d7.3H), 0.96 (d7.3H), 1.05 (d7.3H), 1.52 (s, 3H) , 1.70 (s, 3H), 1.87 (s, 3H), 3.25 (m, 2H), 3.70 (dl 1.1H), 3.94 (d6, 1H), 4.28 (d6, 1H), 5.06 (t8.1H) and 5.20 (d10, 1H)

IR (Nujol): 1720, 3450 cm ^-1 .

The mass scale shows M ⁺ ion at m / z 600; as well as fragment ions at 622, 604, 579, 517,499, 400, 356, 263 and 95 m / z. The combined ethyl acetate extract was evaporated, the residue was taken up in about 100 ml of methanol, the resulting suspension was filtered and the filtrate was evaporated to dryness.

The methanol extract was evaporated, the oily residue dissolved in water and extracted with ethyl acetate. The combined ethyl acetate extract was added to the residue from the supernatant and the resulting mixture was extracted. The combined ethyl acetate extract was added to the residue from the supernatant and the resulting mixture was dried.

The residue was dissolved in about 30 ml of chloroform / ethyl acetate (3: 1) and loaded onto a 100 ml silica gel column (70-230 mesh) containing sand. We collect 20 mles fractions, 11-32. fractions were combined and evaporated to dryness. The residue was purified by preparative HPLC on a Spherisorb S5 ODS-2 column using a 1: 1 mixture of acetonitrile and water at a flow rate of 30 mL / min and detected at 238 nm. The peaks between 29.6 and 31.6 minutes were eluted, combined, diluted with equal volume of water, pumped back to the top of the column and eluted with acetonitrile. The eluate was evaporated, the residue dissolved in acetone and cyclohexane and lyophilized. 42.4 mg of the title compound are obtained.

1 H-NMR (CDCl 3, ppm, 250 MHz): 0.79 (d7.3H), 0.96 (d7.3H), 1.06 (d6.3H), 1.30 (s, 3H) , 1.60 (s, 3H), 1.69 (s, 3H), 1.89 (s, 3H), 3.29 (m, 1H), 3.75 (d10, 1H), 3.98 ( d6, 1H), 4.30 (t6.1H), 5.41 (s, 1H), 5.66 (d15, 1H).

The mass pattern shows M ⁺ ions at m / z 628 and fragment ions at m / z 610, 592, 574,484,441,423, 370, 334, 316, 265, 247, 237, 219 and 167.

Example 3

Preparation of 12-desmethyl-23 (E) - (methoxyimino) - (Factor A) -12-carboxylic acid

The product of Example 2 was stirred with 12.5 mg of methoxyamine hydrochloride and 20.7 mg of sodium acetate in 0.5 ml of methanol for 16 hours, then diluted with dichloromethane, washed with water (25 ml) and brine. After evaporation of the organic phase, the foamy residue was dissolved in a mixture of chloroform, methanol and acetic acid (180: 8: 1) and poured onto a column of silica gel (Merck Kieselgel 60), 230 g to 400 mesh. Elution with the above solvent system gave 62 mg of the title compound, [α] θ = + 49 ° (c = 0.4, in chloroform).

? ^{H is} 248.2 nm (e _max 26000), y _max (CHBr ₃ ) 2470 (OH), 1740 (CO ₂ H) and 1708 cm -1 (lactone);

¹-NMR (CDCl ₃ , δm): 0.92 (d, 6Hz, 3H), 0.97 (d, 6Hz, 3H), 1.07 (d, 6Hz, 3H), 1.87 (s , 3H) 3.2-3.4 (m, 3H), 3.83 (s, 3H), 3.96 (d, 6Hz, 3H) and 4.29 (d, 6Hz, 3H).

Example 4

Preparation of 12a-Hydroxy-23-oxo (Factor A) ml A medium in a 250 ml shake flask Streptomyces platensis subsp. Malvinus was directly inoculated with NRRL 3761 (ATCC 29778) and incubated at 250 rpm in a rotary shaker at 250 rpm for 2 days. This 2-day culture was transferred to 5 ml aliquots in 50 ml shake flasks and a 50 µl 23-oxo (Factor A) compound in methanol (50 mg / ml) was added, resulting in 500 mg / l. The fermentation was continued under the same conditions for 3 days, followed by the addition of an equal volume of methanol to the shaking flasks and shaking for 1 hour before centrifugation.

The supernatant was evaporated to dryness and the residue was extracted with a mixture of 0.05 M ammonium acetate solution adjusted to pH 4.5 with acetonitrile and acetic acid. The extract was clarified and the supernatant was fractionated in 200 µl portions on a Spherisorb S5 ODS-2 column of 100 x 4.6 mm. Detection was performed at 238 nm. As eluent, a gradient from 40 to 60 to 60:40 of acetonitrile / 0.05 M ammonium acetate solution at pH 4.5 is used at a constant flow rate of 3 ml / min. After each injection, the peak after 4.3 minutes was eluted and collected. All such fractions were combined and evaporated. The title compound is thus obtained as a solid.

The mass pattern shows M ⁺ ions at m / z 626 and fragment ions at m / z 608,565,498,455,437,386 and 167.

Example 5

Preparation of 12a-Hydroxy (Factor A)

2.5 g of factor A dissolved in 50 ml of methanol in Example 1

EN 213 349 (ATCC 23934), then fermentation continued under the same conditions for a further 5 days, and the cells were separated by centrifugation. The cells were mixed with methanol and centrifuged after 30 minutes. This gave 900 ml of methanol extract. The supernatant of the culture fluid was extracted with ethyl acetate, and the combined ethyl acetate extract was evaporated and the residue was dissolved in methanol extract of the cells. The resulting mixture was evaporated, the residue dissolved in 200 ml of methanol, poured onto a 125 cm * 5 cm Sephadex LH20 column and eluted with methanol.

ml fractions are obtained, see pages 47-57. fractions were combined and evaporated. The residue was dissolved in a 1: 1 mixture of acetonitrile and water (10 mL) and subjected to preparative pressure chromatography on a 250 mm * 20 mm Spherisorb 5S ODS-2 column. The elution was carried out with a 1: 1 mixture of acetonitrile and water at a constant flow rate of 20 ml / min and detected at 250 nm. The column was charged with 1.5 ml portions, collecting the peak eluate between 14.8 and 15.6 minutes, diluting with an equal volume of water, refluxing to the top of the column and eluting with acetonitrile. Acetone and cyclohexane are added to the residue of the acetonitrile solution and lyophilized. 25.1 mg of the title compound are obtained as a colorless solid.

IR spectrum (CHBr ₃ ): 996, 1710 and 3500 cm ^-1 , ¹³ C-NMR (62.5 MHz): 10.8 (q), 13.7 (q), 15.4 (q), 19.7 (q), 22.7 (q), 26.7 (d), 34.7 (t), 35.9 (d), 36.0 (t), 40.6 (t), 41 , 0 (t), 44.3 (d), 45.5 (d), 66.2 (t), 67.5 (d), 67.7 (d), 68.3 (t), 69, 1 (d), 76.6 (d), 79.2 (d), 80.1 (s), 99.6 (s),

117.8 (d), 119.7 (d), 120.9 (d), 127.2 (d), 130.5 (s), 135.9 (s), 137.1 (d), 137.3 (d), 137.8 (s), 140.9 (s) and 173.2 (s);

Β-NMR spectrum: the same as that of the product obtained in Example 6.

Example 6

Preparation of 12a-Hydroxy (Factor A) and 4a, 12, α-dihydroxy (Factor A)

A solution of 2.5 g of factor A in 50 ml of methanol was used

Streptomyces rimosus NRRL 2455 (ATCC 14827) obtained by the method described in Example 1 was fermented under the same conditions for 5 days and the cells were removed by centrifugation. The supernatant is discarded, the cells are washed with water and centrifuged again. The aqueous wash was discarded, the cells were extracted with methanol and centrifuged. This gave 1300 ml of methanol extract which was evaporated. The oil-like residue was extracted with 250 ml of acetonitrile and 200 ml of methanol, and the extracts were combined and evaporated. The oil-like residue was dissolved in 200 ml of methanol and fractionated in 50 ml portions on a 130 cm ^x 5 cm Sephadex LH20 column. Methanol was used as eluent. 26-36. fractions were combined, evaporated, and the residue was dissolved in a mixture of 15 ml of acetonitrile and water (55:45).

1.8 ml portions of preparative pressurized liquid chromatography (hereinafter HPLC) on a 250 nm> 20 mm spherisorb S5 ODS-2 column eluting with acetonitrile / water 55: 45 (0-12.5 min) followed by acetonitrile and a 70: 30 mixture of water (12.5 to 21.0 minutes) and finally acetonitrile (21.0 to 30 minutes) at a flow rate of 20 ml / min. The column was then equilibrated with 55: 45 acetonitrile / water. The fractions eluted between 14.8 and 15.5 minutes were collected, pooled and refluxed to the top of the column. The column was eluted with acetonitrile and the residue obtained after evaporation of acetonitrile was lyophilized after addition of acetone and cyclohexane. 32.3 mg of 12α-hydroxy (factor A) are obtained as a colorless solid.

1 H-NMR (CDCl ₃ , δppm, 250 MHz): 0.80 (d, 3H), 0.96 (d, 3H), 1.06 (d, 3H), 1.88 (s, 3H) ), 3.27 (m, 1H), 3.40 (t9, 1H), 3.74 (dl, 1H), 3.96 (d6, 1H), 4.30 (lake, 1H), 5, 02 (m, 1H) and 5.21 (d9.1H).

The mass pattern shows M ⁺ ions at m / z 628 and fragment ions at m / z 610, 592,482,464, 441, 423, 370, 330, 297,265, 247,237, 219, 167 and 95.

Similarly, 26.4 mg of 4a, 12a-dihydroxy (Factor A) were obtained from the fractions eluted between 6.2 and 6.8 minutes as a colorless solid.

1 H-NMR (CDCl 3, δppm, 250 MHz): 0.80 (d6.3H), 0.96 (d6.3H), 1.06 (d6.3H), 1.56 (s, 3H) , 3.31 (m; 1H), 3.40 (ddll, 9; 1H), 3.75 (d10; 1H), 3.97 (d6; 1H), 4.24 (dl4; 1H), 4, 33 (dl4; 1H), 4.58 (d6; 1H) and 5.72 (s; 1H).

The mass spectra show M ⁺ ions at m / z 644 and fragment ions at m / z 626, 608,482,464,457,439,297, 265, 247, 237, 219 and 167.

Example 7

Preparation of 12-Hydroxy (Factor A)

A solution of 2.5 g of factor A in 50 ml of methanol was added to the culture of Absidia cylindrosporaNRRL 2796, prepared by the method described in Example 1, except that the following composition was used:

g / L

Corn Soaking Liquid 20.0

Meritose 10.0

Soy Oil 1.0

Distilled water as needed

The pH was adjusted to 4.8-5.0 before autoclaving with potassium hydroxide.

The fermentation was continued under the same conditions for a further 5 days, then the cells were removed by centrifugation and the cells were extracted with 400 ml of methanol.

About 900 ml of the supernatant of the culture fluid was evaporated and the residue was extracted with ethyl acetate.

Claims (5)

Patent Claim Point A process for the preparation of a compound of formula (I) and salts thereof wherein R is methyl, hydroxymethyl or carboxyl; HU 213 349 Β R ¹ is C 1-4 alkyl, R ² is hydroxy when R ³ is hydrogen R ² and R ³ together with the carbon atom to which they are attached represent a group of the formula> C = O or> C = NOR ⁹ - wherein R ⁹ is a C 1 -C 1 alkyl group and the group> C = NOR ^{9 is in} the E configuration . R ⁴ is hydroxy or C 1-4 alkoxy, R ⁶ is hydrogen or hydroxy, with the proviso that when R ⁶ is hydrogen, R is hydroxymethyl or carboxyl, further when R ⁶ is hydroxy, R is methyl and R ⁷ is hydrogen or hydroxy, wherein a compound of formula ² wherein R ¹ , R ² , R ³ and R ⁴ are as defined herein is capable of selectively oxidizing Streptomyces of formula 2 or an Absidia microorganism or an enzyme derived therefrom or an enzyme derivative thereof or a preparation containing said enzyme and optionally for the preparation of compounds of the formula (I) wherein X is> C = NOR ^{9 the} corresponding 23-oxo reacting the compound with H ₂ NOR ⁹ , wherein R ⁹ is as defined above, and optionally converting the resulting compound (1) to a salt. A process for the preparation of a compound of formula (I) according to claim 1 wherein R ¹ is methyl, ethyl or isopropyl and R ⁴ is hydroxy or methoxy, characterized in that the appropriate starting materials are used. A process for the preparation of a compound of formula (I) according to claim 1, wherein R ¹ is isopropyl and R ⁴ is hydroxy or methoxy, characterized in that the appropriate starting materials are used. A process for the preparation of a compound of formula (I) according to claim 1, wherein R is a carboxyl group, characterized in that the appropriate starting materials are used. A process for the preparation of a compound of formula (I) according to claim 1 wherein R ² and R ³ together form a carboxyl group R> C = NOCH ₃ and R ^{4 a} hydroxy group, characterized in that the appropriate starting materials are used.