HU210561B - Process for producing 9-alpha-hydroxi-3-oxo-4,24(25)-stigmadien-26-acid derivatives - Google Patents

Abstract

The present invention relates to a compound of formula (I) wherein M is hydrogen, C1-C4 alkyl or a pharmaceutically acceptable cation for the preparation of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid derivatives. comprising a steroid mixture of β-sitosterol or a plant derived β-sitosterol from Mycobacterium sp. The microorganism strain BCS-394, designated NCAIM B (P) 001 038, has undergone downward cultivation under aerobic conditions on an assimilable carbon- and nitrogen source medium, in the presence of mineral salts, from the fermentation broth containing M and / or methyl (M). The compound (I) or compounds of formula (I) are optionally isolated and optionally converted to the compound of formula (I), wherein M is hydrogen, to form a compound of formula (I) having a pharmaceutically acceptable cation or C 1-4 alkyl group. The invention also relates to a method for the preparation of pharmaceutical compositions containing the above novel steroid derivatives as active ingredients. OH («·) HU 210 561 Scope of the description: 8 pages (including 1 sheet)

Description

The present invention relates to a process for the preparation of 9-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid derivatives of the formula (I) wherein M represents a hydrogen atom, a C 1 -C 4 alkyl group or a pharmaceutically acceptable cation. microbiological degradation and, if appropriate, subsequent salt formation or esterification.

The compounds of Formula I containing a pharmaceutically acceptable cation M include salts with inorganic and organic cations such as sodium, potassium, calcium, magnesium, aluminum, zinc, ammonia, ethylenediamine or tris / hydroxymethyl. a cation derived from aminomethane and tetramethylammonium ion.

The compounds of formula (I) according to the invention are novel compounds and have valuable therapeutic efficacy. In addition, they serve as starting materials for the preparation of further pharmaceutically valuable steroid derivatives.

Bacteria utilizing sterols as carbon sources are known to degrade both the sterane skeleton and the side chain. The mechanism of sterol side chain degradation has been discovered by C. J. Sih et al. Chem. Soc. 89, 1957 (1967), 104, 4718 (1982), 104, 4720 (1982)]. The degradation of the cholesterol side chain begins with end-chain hydroxylation. Oxidation of the hydroxyl group on the 26 carbon atoms produces a carboxylic acid, which is degraded by the β-oxidation mechanism characteristic of fatty acids and by the gradual shortening of the side chain. The degradation of the side chain of β-sitosterol substituted on ethyl carbon at C24 differs from cholesterol in that the chain-breaking bacterium forms a carboxyl group on carbon 28 attached to carbon 24 and then cleaves it by a β-oxidation mechanism. and a bond between the 28 carbon atoms. In parallel with the chain breakdown, enzymatic changes in the sterane backbone lead to the breakdown and subsequent complete degradation of the backbone. The skeleton is broken down to form 9a-hydroxy-1,4-diene-3-one steroid derivatives of the 3β-1ύύΓθχί-5-έη structure and to spontaneously rearrange it into pharmaceutically valuable secosteroids containing the B ring of water. it opened between the 9 and 10 carbon atoms. [R. M. Dodson and R. D. Muir, J. Am. Chem. Soc., 83, 4627 (1961), K. Schubert et al., Z. Naturforsch. 15b, 584 (1960)].

In the 1970s, mutation-based strains that did not contain enzymes that catalyze the degradation of the steroid skeleton were produced by genetic methods based on mutation from sterol-utilizing bacteria.

Bacteria are also known to be completely or partially removable under cell wall enzymes and protoplasts under iso-osmotic conditions [J. Tomcsik and S. Guex-Holzer, Schweiz. Z. Barely. Path, Bact. 75, 517 (1952); C. Weibull: J. Bacteriol. 66, 688 (1953)] and spheroplasts [H. Sato et al., Canad. J. Microbiol. 77, 807 (1965)], which can be transformed into intact cells by regeneration of the cell wall, and polyethylene glycol-induced fusion of protoplasts or spheroplasts with genetic recombination events [K. Fodor and Alföld L., Proc. Natl. Acad. Sci. USA 73, 2174 (1976); P. Schaeffer et al., Proc. Natl. Acad. Sci. USA 73, 2151 (1976); N. Rastogi et al., J. Gene. Microbiol. 729, 1227 (1983), 137a, 135 (1986)].

In our research, the method developed by G.E. Peterson et al. Lipid Research 3, 275 (1962)] isolated sitosterol-degrading microorganisms from various soil samples. A strain of Mycobacterium utilizing this sterol utilizing microorganism was selected from which mutant strain no longer disrupts the sterane skeleton by ultraviolet irradiation. From this strain, mutant strains genetically labeled with mutagenic treatment with N-methyl-N'-nitro-N-nitrosoguanidine, such as strains requiring various nutrients (e.g., adenine, proline, valine) and resistant to various antibiotics (e.g., streptomycin), were prepared. In vivo genetic recombination of these genetically labeled mutant strains by spheroplast fusion yielded recombinant mycobacterial strains that partially cleave the sterol side chain. Such a Mycobacterium strain, BCS 394, which is a major component of sitosterol, has not been previously described in the literature as a major degradation product, namely 9α-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid (I, where M). is hydrogen) and produces 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid methyl ester (I where M is methyl), - NCAIM B (P) 001 083 20 January 1988 was deposited in the National Collection of Agricultural and Industrial Microorganisms (Budapest).

The taxonomic characteristics of the tested NCAIM B (P) 001 038 strain Mycobacterium strains are as follows: colonies of the microorganism are off-white, grow at 45 ° C, do not grow at 50 ° C and cannot grow in the absence of oxygen. It sells ammonium chloride, calcium nitrate, sodium nitrate and ammonium hydrogen phosphate as its only N source. It uses glucose, glycerol, fructose, maltose, xylose, sodium succinate, sodium pyruvate, sodium acetate as the only source of carbon, and does not sell sucrose, starch, arabinose. The strain is not capable of tyrosine degradation and has no pigmentation.

Further, it has surprisingly been found that the novel compounds of formula I, where M is as described above, are very valuable in inhibiting serum cholesterol lowering effects on mammalian cholesterol biosynthesis.

Epidemiological studies in the 1980s made it clear that high serum cholesterol levels are one of the most important risk factors for the development of coronary heart disease. The major organ of cholesterol formation is the liver, the biosynthesis starts from acetic acid and leads to cholesterol via mevalonic acid and then terpene-structured squalene [K. Block: Angew. Chem. 77, 944 (1965), F. Lynen, Angew. Chem. 77, 929 (1965)].

HU 210 561 A

Among the active ingredients that inhibit cholesterol biosynthesis, 8 (14) -dehydro-15-oxo-sterol derivatives occupy an important place, the most effective of which has been shown to lower the level of 3β-hydroxy-5α-cholesterol in several animal species and humans. [G. J. Schroepfer, jr. et al., J. Bioi. Chem. 263, 4110 (1988); G. J. Schroepfer, jr. et al., Proc. Nat. Acad. Sci. (USA) 79, 3042 (1982)].

The inhibition of cholesterol biosynthesis by 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid derivatives of the present invention is provided by 9a-hydroxy-3-oxo4,24 (25) -stigmastadiene-26-acid (I, wherein M is hydrogen). The inhibitory activity of this compound by the action of 33-hydroxy-5α-cholest-8 (14) -en-15-one (Colestolon) according to the method of A. Endo et al., Eur. J. Biochem. 77, 31 (1977); Biochim. Biophys. Acta, 486, 71 (1977)], examining the incorporation of radioactive acetic acid into cholesterol in rat liver homogenate. Male Hannover Wistar rats weighing 50-60 g were used for the experiments. The animals were housed in a 5% cholestyramine-enriched rat diet for 1 week prior to the experiment in an inverted light cycle (16h to 4h in the light, 4h to 16h in the dark). The animals received food and water ad libitum.

On the day of the experiment, the animals were decapitated, the livers were placed in ice-cold Krebs / Ringer bicarbonate buffer (pH 7.4) and cut into small pieces. Tissue homogenizers were used to prepare very homogeneous coarse homogenates and weighed 100 mg wet liver homogenates into glass stoppered tubes. The operations were performed under constant cooling. The incubation mixture in the liver homogenate, the Krebs / Ringer bicarbonate buffer, and l- ^l4 C sodium acetate (manufactured IZINTA, specific activity 14.86 MBq / mg) and the ³ H-cholesteryl oleate (manufactured by Dupont, specific activity: 3067 GBq / mmol). The steroids which inhibit cholesterol biosynthesis were added to the incubation mixture in an alcoholic solution prepared by GJ Schroepfer et al. The final volume of the incubation mixture was 2.0 ml. Incubation was continued for 2 hours in a 37 ° C water bath. After 2 hours, 2 mL of 15% alcoholic KOH was added and kept at 7075C for another 2 hours. Lipids from the alkaline mixture were extracted with petroleum ether. The extract was evaporated under a stream of nitrogen, the dry residue was dissolved in acetone-ethyl alcohol and cholesterol was precipitated with digitonin solution. The precipitate was centrifuged and dissolved in a scintillation mixture and its activity was measured on a Hitachi scintillation spectrometer. Activity was measured for 5 minutes. The results of this study are summarized in Table 1.

Table 1

Compound Concentration μΜ Inhibition% 3 P-hydroxy-5a-cholest 2.5 16.6 8 (14) -en-15-on (reference) 10 55.0 9a-hydroxy-3-oxo-4,24 (25) - 2.5 65.3 stigmastadiene-26-acid (I) 10 86.3

The data in Table 1 indicate that the compound of the present invention significantly inhibits the cholesterol biosynthesis inhibitory activity in the rat liver liver homogenate assay by the known inhibition of 3P-hydroxy-5α-cholest-8 (14) -en-15-one (Colestolon).

SUMMARY OF THE INVENTION The present invention relates to a process for the preparation of 9a-hydroxy-3-oxo-4,24 (25) stigmastadiene-26-acid derivatives of the general formula (I) wherein M represents a hydrogen atom, a C 1 -C 4 alkyl group or a pharmaceutically acceptable cation. comprising preparing a sterol mixture containing β-sitosterol or a plant-derived β-sitosterol with Mycobacterium sp. BCS-394 is immersed under aerobic conditions in immersed culture of strain NCAIM B (P) 001 038 on an assimilable carbon and nitrogen source in the presence of mineral salts, the resulting fermentation broth having M at the H and / or methyl group. The compound (s) of formula (I) is optionally isolated and optionally the compound of formula (I) wherein M is hydrogen is converted to the compound of formula (I) wherein M is a pharmaceutically acceptable cation or C 1-4 alkyl.

In a preferred embodiment of the method of the invention, the Mycobacterium strain NCAIM B (P) 001 038, BCS-394, is cultured on media containing assimilable carbon and nitrogen and inorganic salts suitable for the cultivation of rapidly growing Mycobacteria. Of the carbon sources, glucose and glycerol, organic nitrogen sources, soy flour, urea, yeast extract, and inorganic nitrogen sources, ammonium chloride, are preferred. The sterol to be converted is preferably added to the medium in the form of a suspension in polypropylene glycol and Tween 80. The substrate for microbiological conversion is either pure β-sitosterol or a sterol mixture containing β-sitosterol, such as crude sitosterol obtained from soybean oil, which is a mixture of β-sitosterol and campesterol.

The cultivation of the microorganism and the sterinate conversion is carried out at 28 to 37 ° C, preferably at 32 ° C.

The products of the sterinate conversion are bound to a greater extent to mycobacterium cells and to a lesser extent to the culture medium. The steroids are extracted from the cells with water-miscible organic solvents, preferably methanol, and the products formed during the sterol transformation are conveniently isolated from the filtrate of the fermentation broth by extraction with ethyl acetate or chlorinated hydrocarbons such as dichloromethane. The crude product obtained by evaporation of the extracts usually contains several sterol degradation products, in addition to 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid and its methyl ester. Column chromatography and / or thin layer chromatography may conveniently be employed to isolate the sterolization products. The structure of the products was determined by UV, IR, NMR and ⁱ³ * C-NMR and mass spectroscopic methods.

HU 210 561 A

The compound of formula I prepared by the process of the invention, wherein M is hydrogen, may optionally be formulated as a salt for pharmaceutical use. These salts can be prepared in a manner known per se from the compound of formula (I) wherein M is hydrogen and the base providing the corresponding cation.

The compound of formula (I) wherein M is hydrogen may be prepared from the compound of formula (I) where M is hydrogen, if desired, in a manner known per se, where M is a C 1-4 alkyl group. Ester derivatives where M in the formula (I) is methyl may also be isolated directly from the fermentation broth.

The compounds of formula (I) wherein M is hydrogen, (C 1 -C 4) -alkyl, or a pharmaceutically acceptable cation may be advantageously used in humans as active ingredients in antihypercholesterolemic drugs, for example in the treatment of atherosclerosis or hyperlipaemia. They may be administered, for example, orally or parenterally, for example in the form of capsules, tablets or injectable formulations. In general, oral administration is preferred. The dosage will, of course, vary depending on a number of factors, such as the age, weight, severity of the condition and other factors of the subject being treated, but in general the daily dosage for adults will be about 100 mg to 3000 mg, preferably 300 mg to 1000 mg. However, higher dosages may be advantageously employed if necessary.

For therapeutic use, the active compounds of the present invention are conveniently formulated into pharmaceutical compositions by admixing them with carriers and / or excipients suitable for non-toxic, inert, solid or liquid enteral or parenteral administration, as is commonly used in pharmaceutical formulations.

According to the present invention, solid pharmaceutical forms for oral administration can be prepared from the following compounds of formula (I) wherein M is the above-9a-hydroxy-3-oxo-4,24 (25) stigmastadiene acid:

The active ingredient powder having an average particle size of 1.0-0.05 mm is homogenized with similar particle size excipients used in tablet and capsule formulation, and the powder mixture is either directly or dry or after wet granulation it is filled into gelatin capsules or compressed into tablets.

As excipients, fillers used in the manufacture of tablets and capsules, preferably starch, tricalcium phosphate, calcium hydrogen phosphate, lactose, glucose, cellulose, sucrose, mannitol, sorbitol or a mixture thereof; adsorbents, preferably colloidal silica (protected under the name Aerosil, Gesilite, Cab-o-sil, Syloid, Neosyl); binders, preferably gelatin, gum arabicum, cellulose ethers (methylcellulose, carboxymethylcellulose), pectin, sodium alginate, tragacanthate, polyvinylpyrrolidone, polyvinyl alcohol or a mixture thereof, disintegrating agents, starch, formaldehyde casein, pectin, bentonite, microcrystalline cellulose or a mixture thereof, glidants, lubricants and anti-adhesives, preferably metal soaps, magnesium and calcium stearate), glycerol monostearate, sterile alcohol, cetyl alcohol, polyethylene alcohol, polyethyl alcohol, polyethyl alcohol, polyethyl alcohol, polyethyl alcohol, polyethyl alcohol.

The process of the present invention is illustrated by the following examples:

Example 1

Mycobacterium sp. A 5-ml culture suspension of BCS-394 (NCAIM B (P) 001 038) grown on potato-glucose slant agar was prepared in 10 ml sterile water and inoculated with 800 ml MI in a sterilized 800 ml in a 3000 ml Erlenmeyer flask. . The composition of MI medium is as follows:

glucose 8.0 g soybean meal 1.6 g urea 0.4 g yeast extract 0.8 g ammonium chloride 2.4 g potassium dihydrogen phosphate 0.4 g magnesium sulphate 7H ₂ O 0.4 g iron (III ) -chloride-6H ₂ O 0.4 g

Tween 80 0.4 g

800 ml tap water

The medium was adjusted to pH 7.0 prior to sterilization and sterilized at 121 ° C for 45 minutes.

The flask was shaken at 32 ° C on a mill sieve shaker (250 rpm, 2.5 cm displacement) for 2 days, and the resulting inoculum culture was sterilized with 4.2 liters of 4.2 L at 121 ° C for 4.2 minutes. MF medium was inoculated.

MF was prepared as follows: Glucose g, 10 g Soya flour, 2.5 g Urea, 15 g Ammonium chloride and 2.5 g Potassium dihydrogen phosphate were boiled in 1.5 L of tap water. To this basic medium, supplemented with 15 g of calcium carbonate, 100 g of β-sitosterol, 40 g of polypropylene glycol, 10 g of Tween 80 and 800 ml of tap water are added and the mixture is constantly stirred for one hour at 121 ° C. and then make up to 4 liters with hot tap water. The medium thus obtained is then sterilized for one hour at 121 ° C.

The culture obtained by inoculating the MF medium was stirred at 32 ° C and 750 rpm with 100 L of air for 24 hours. The fermentation is then continued for 6 days by introducing 300 liters of air per hour into the 750 rpm culture. After the fermentation is stopped, the products of the sterol conversion are obtained as follows.

of fermentation broth containing 20 g of β-sitosterol at the start of the fermentation, the cells are separated by filtration, from which they are extracted three times with 200 ml of methanol.

Dissolve the sterol degradation products and the unmodified starting material. The filtrate of the fermentation broth was extracted with 200 ml of ethyl acetate. The combined methanol and ethyl acetate extracts were evaporated in vacuo to give 22 g of crude product which was chromatographed on a column of 400 g of silica with gradually increasing ethyl acetate-n-heptane mixtures. Fractions soluble in 10% ethyl acetate in ethyl acetate-n-heptane were evaporated and the residue was recrystallized from methanol to give 0.15 g of 4-stigmasten-3-one.

M.p. 89-90 'C;

UV spectrum (EtOH):? _Max 243 nm (ε - 14,090); Infrared (KBr): vC = O 1678, vC = C

1616 cm ^-1 ;

1 H NMR (CDCl ₃ , δ): 5.56 s (H-4), 1.16 s (3

H-19), 0.68 s (3H-18) ppm;

Mass Spec: Molecular ion (m / z): 412;

Typical ions (m / z): 412.229, 135, 124,109.95, 55.43.

A mixture of 20% ethyl acetate in ethyl acetate-n-heptane dissolves 1.05 g of the unmodified β-sitosterol campesterol mixture in the column.

Fractions soluble in 30% ethyl acetate / ethyl acetate / n-heptane were evaporated in vacuo and the residue was recrystallized from methanol to give 1.8 g of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid methyl ester. we get.

Mp 187-189 ° C;

UV spectrum (EtOH):? _Max 239 nm (ε = 25530); Infrared spectrum (KBr): vOH 3600-3400, max. 3487 (9a-hydroxyl group), v C = O 1717 (conjugate ester), 1678 (ketone 3), v C = C 1615 cm ^-1 .

1 H NMR (C ₆ D ₆ , δ): 5.92 s (H-4), 3.46 (3 H,

OCH ₃ ), 2.58 q (2 H-28), 1.96 s (3 H-27), 1.22 t (3

H-29), 0.95 (3 H-21), 0.8 s (3 H-19), 0.55 s (3 H 18) ppm;

Mass Spec: Molecular ion (m / z): 470;

Typical ions (m / z): 470, 438, 410, 137, 136, 124.

Fractions soluble in 35% ethyl acetate in ethyl acetate-n-heptane were evaporated in vacuo and crystallized from methanol to give 0.15 g of 9a-hydroxy-27-nor-4-cholesterol-3,24-dione.

M.p. 208-211 ° C;

Ultraviolet Spectrum (EtOH): 241 nm (ε = 14,800);

Infrared (KBr): vOH 3555, v C = O 1705, (ketone 24), 1660 (ketone 3), v C - C 1-14 cm ^-1 ; 1 H NMR (CDCl ₃ , δ): 5.86 s (H-4), 1.32 s (3

H-19), 1.05 t (3 H-26), 0.90 d (3 H-21), 0.72 s (3

H-18) ppm;

Mass Spec: Molecular ion (m / z): 400;

Typical ions (m / z): 400, 151, 137, 136, 124, 122,

109.57.

Fractions soluble in 40% ethyl acetate in ethyl acetate-n-heptane were evaporated and crystallized from acetone to give 0.2 g of 9a-hydroxy-3-oxo-23,24-dinor-4-cholene-22-acid methyl ester.

M.p. 212-216 'C;

Ultraviolet _Spectrum (EtOH): λ _ηΰχ 242 nm (ε = 15,860);

Infrared (KBr): vOH 3395, v C = O 1730 (ester), 1650 (ketone 3), ZnC = C 1615 cm ^-1 ;

1 H NMR (CDCl ₃ , δ): 5.8 s (H-4), 3.6 s (OCH ₃ ), 1.3 s (3 H-19), 1.2 d (3 H 21), 0.7 s (3

H-18) ppm;

Mass spectrum: Molecular ion (m / z): 374;

Typical ions (m / z): 374, 180, 151, 137, 136, 124,

109.81;

Fractions soluble in 42% ethyl acetate in ethyl acetate-n-heptane were evaporated and recrystallized from methanol to give 0.5 g of 3-oxo-4-stigmastenoic acid.

Mp: 164-168 ° C;

Ultraviolet Spectrum (EtOH): 242 nm (ε =

010);

Infrared (KBr):? C = O 1736, 1650 (ketone 3 and acid 26),? C = C 1616 cm ^-1 ;

1 H NMR (CDCl ₃ ): 5.73 s (H-4), 1.19 s (3

H-19), 0.7 s (3H-18) ppm;

Mass Spec: Molecular ion (m / z): 442;

Typical ions (m / z): 442,424,229,147,124,123,95,55;

The fractions soluble in 45% ethyl acetate / ethyl acetate / n-heptane were evaporated in vacuo to give 2.5 g of a residue which was dissolved in 10 ml of methanol, 10 ml of 10% methanolic hydrochloric acid and 50 ml of ethyl acetate. we add. The resulting solution was washed with water (2 x 30 mL) and evaporated in vacuo. The residue was recrystallized from methanol to give 2.2 g of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid.

M.p. 242-248 'C;

Ultraviolet _Spectrum (EtOH): λ _ΜΧ 239 (ε - 21 230); Infrared spectrum (KBr): vOH 3650-3350, max. 3483 (9a-hydroxyl group), 3200-2200 (acid 26), vC = 0 1673 (ketone 3 and acid 26), vC = C

1614 cm ^-1 ;

1 H NMR (C ₅ D ₅ N, δ): 6.15 d (H-4), 2.83 q (2

H-28), 2.28 s (3 H-27), 1.39 s (3 H-19), 1.39 t (3

H-29), 1.14 d (3H-21), 0.89 s (3H-18) ppm; Mass Spec: Molecular ion (m / z): 456;

Typical ions (m / z): 45.6 438, 410, 137, 136, 124,

123, 110.

The resulting 9a-hydroxy-3-oxo-4,24 (25) -stigmadiene26-carboxylic acid is obtained according to Houben-Weyl: Methoden der Org. Chem. VIII. Volume 516-526. (E. Müller Verlag, Thübingen, 1952) can be easily converted to the methyl, ethyl or propyl ester.

Example 2

Preparation of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid and its methyl ester

AMycobacterium sp. A culture suspension of BCS-394 strain cultured on potato glucose slant agar for 4-5 days was prepared in 10 ml of sterile water and 100 ml of 100 ml of sterile MY-5 medium was inoculated in 1 ml of 10 ml 500 ml Erlenmeyer flasks. Prepare MY-5 medium as follows:

g of glycerol, 1 g of soy flour, 0.5 g of urea, 1 g of ammonium chloride, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulphate 7H ₂ O, 1 g of sodium citrate and 0.05 g of iron (III). ) -chloride x 6H ₂ O is boiled in 300 ml of tap water and then 30 g of crude sitosterol (2: 1 β-sitosterol-campesterol) are added.

The suspension), containing 8 g of propylene glycol, 2 g of Tween 80 and 150 ml of tap water, sterilized at 121 ° C with stirring to a sitosterol melt, and finally the suspension containing the media components is made up to 1 liter with hot tap water.

The flasks were shaken at 32 ° C on a flat shaker (250 rpm, 2.5 cm displacement) for 7 days. The cells are then separated from the fermentation broth by centrifugation, from which the sterol degradation products and the starting material are dissolved in three times 200 ml of methanol. The methanol eluate was evaporated in vacuo. The filtrate of the fermentation broth was extracted with 200 ml of ethyl acetate and the extract was concentrated in vacuo. The evaporation residues of the extracts were combined to give 32 g of crude product which was chromatographed on a 500 g silica gel column with gradually increasing mixtures of ethyl acetate with n-heptane-ethyl acetate.

2.5 g of unchanged starting material (2: 1 mixture of β-sitosterol-campesterol) are dissolved in the column with 25% ethyl acetate in n-heptane-ethyl acetate.

Fractions soluble in 30% ethyl acetate containing n-heptane-ethyl acetate were evaporated in vacuo and the residue was recrystallized from methanol to give 3.4 g of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid methyl ester. .

M.p .: 187-189 'C.

The product has the spectroscopic characteristics shown in Example 1.

Fractions soluble in 35% ethyl acetate in n-heptane-ethyl acetate were evaporated in vacuo and the residue was recrystallized from methanol to give 0.43 g of 9a-hydroxy-27-nor-4-cholestene-3,24-dione.

Mp 208-211 'C.

The product has the spectroscopic characteristics shown in Example 1.

The chromatographic fractions dissolved in 37% ethyl acetate containing n-heptane-ethyl acetate were evaporated and the residue was recrystallized from acetone to give 0.49 g of 9a-hydroxy-3-oxo-23,24-dinor-4-cholene-22-acid methyl ester. win.

Mp: 212-216'C.

The product has the spectroscopic characteristics shown in Example 1.

Fractions soluble in 40% ethyl acetate in n-heptane-ethyl acetate were evaporated and the residue was recrystallized from methanol to give 0.25 g of 9-hydroxy-26,27-dinor-4-cholestene-3,24-dione. M.p .: 201-204 'C;

Ultraviolet Spectrum (EtOH): 241 nm (ε = 14,200);

Infrared (KBr): vOH 3550, v C = O 1705 (ketone 24), 1660 (ketone 3), v C = C 1610 cm ^-1 ; 1 H NMR (CDCl ₃ , δ): 5.85 s (H-4), 2.12 s (3

H-25), 1.3 s (3 H-19), 0.9 d (3 H-21), 0.7 s (3

H-18) ppm;

Molecular ion (m / z): 386;

Typical ions (m / z): 386, 151, 137, 136, 124, 109.43.

Fractions soluble in 45% ethyl acetate in n-heptane-ethyl acetate were evaporated in vacuo. The evaporation residue was purified by preparative thin layer chromatography (developing solvent: ethyl acetate-n-heptane (4: 6)). The product (4 g) was dissolved in methanol (10 ml), acidified with 10% hydrochloric acid in methanol (10 ml) and diluted with ethyl acetate (50 ml). The resulting solution was washed with water (2 x 30 mL) and concentrated in vacuo. The residue was crystallized from methanol to give 3.5 g of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid.

M.p. 242-248 'C;

The product has the spectroscopic characteristics shown in Example 1.

Example 3

Preparation of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid sodium salt

9 mg of 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid is dissolved in 25 ml of ethanol and 2.2 ml of 0.1 M sodium hydroxide is added. The mixture was allowed to stand at room temperature for 30 minutes and then concentrated in vacuo. The oily residue was taken up in 10 ml of ethanol and the solution was evaporated. Acetone (5 mL) was then added and the resulting 9a-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid sodium salt was filtered off. IR spectrum (KBr): vOH 3495, v C = O 1660 (ketone 3), vCOO 1572 cm ^-1 .

Mass Spectrum (FAB): [M + Na] ⁺ = 501.

Claims (2)

PATENT CLAIMS A process for the preparation of 9-alpha-hydroxy-3-oxo-4,24 (25) -stigmastadiene-26-acid derivatives of the general formula (I) wherein M represents a hydrogen atom, a C 1 -C 4 alkyl group or a pharmaceutically acceptable cation. characterized in that the sterol mixture containing beta-sitosterol or a plant-derived beta-sitosterol is characterized by Mycobacterium sp. BCS-394 is fermented under aerobic conditions by immersing a strain of microorganism strain NCAIM B (P) 001 038 in a medium for assimilating carbon and nitrogen in the presence of mineral salts, the resulting fermentation broth having M at the hydrogen atom and / or methyl group. optionally isolating the compound (s) of formula (I), optionally converting a compound of formula (I) wherein M is hydrogen into a pharmaceutically acceptable cation or a (C 1 -C 4) alkyl group of a compound of formula (I). 2. A process for the preparation of a cholesterol lowering medicament comprising the preparation of a compound of formula I according to claim 1, wherein M is as defined in claim 1, alone or without any synergism with the compounds of formula I together with the compound, in a manner known per se in the pharmaceutical formulation.