DE1768985C3 - Process for the production of optically active steroid compounds which contain a 17-hydroxy-8,14-secogona-polyen-14-one nucleus which is alkyl-substituted on the 13 ß-carbon atom (C1 to C4) - Google Patents

Description

a) for the preparation of compounds which have an alkyI-substituted one on the 13/3 carbon atom (Cr to G)) 17j9-hydroxy-8,14-secogona-13.5 (10), 9 (II), 15-pentaen-14-one core contain, compounds which have one on the ^ carbon atom (Cr to C)) - alkyl-substituted

8,14-Secogona-1,3,5 (10), 9 (II) -pentaene-14,17-dione-kern contain the action of an enzyme system from Debaryomyces vini IFO 1214, Pichia wickerhamii IFO 1278, Pichio pijperi IFO 1290, Hansenula capsulata IFO 0984 (NRRL Y-1842) or Hansenula holst «IFO 0980 (NRRL Y-2155), or

b) for the preparation of compounds which have an _{alkyl at the 130-carbon atom (C r} to Gi)

substituted 17 "-hydroxy-8,14-secogona-13.5 ( 10) 3 (11), 15-pentaen- 14-on core included, Compounds which have one on the 13 /? - carbon atom (Ci to C ») - alkyl-substituted

8, l4-Secogona-l33 (l0) 3 (ll), 15-pentaen-14,17-dione-kem contain the action of an E ^ zymsystem von Kloeckera magna IFO 0868 subjects, or

c) for the preparation of compounds containing a 170-hyiiroxy-8,14-secogona-U ^ (10), 9 (II) -tetraen-14-one- core, compounds which have a 8,14-Secogona-13,5 ( \ 0), 9 (11), 15-pentaene-14,17-dione- core, the action of an enzyme system from Candida utiiis IFO 1086, Debaryomyces nicotianae IFO 0855, Pichia etchellsii IFO 128.3, Rhodotorula rubra IFO 0891, Schizosaccharomyces pombe IFO 0362, Hansenula saturnus IFO 0993 or Hansenula holstii IFO.

bond, one substituted on the 13 / l · carbon atom 8,14-Secogona-polyene-14, l 7-dione core contains, one Ring closure reaction to the corresponding 13 /? - carbon-substituted one Gonapolyen-17-one nucleus is subjected to a racemate with respect to the 13-substituents, a yield being obtained which is half the stoichiometric yield at best Furthermore, the steroid of the natural type (in which the 13-substituent is ^ -oriented) is only

ίο obtained by optical splitting.

Furthermore, two chemical processes were used to produce compounds containing one on the 13 /? - carbon atom substituted Ua-Hydroxy-e.H-secogona-13.5 (10), 9 (II) -tetraen-14-one nucleus included, by Miki (one of the inventors) and his coworkers and by C. H. Kuo and coworkers (Chemistry and Industry 1340 (1966)).

However, the compounds obtainable by this process are always 50:50 mixtures of a compound, the one substituted on the 13 /? - carbon atom

The invention relates to a process for the preparation of optically active unsaturated steroids which have a on the 130-carbon atom (Ci- to Ct) -alkyl-substituted 17-Hydroxy-8,14-secogonapolyen · 14-one core (hereinafter referred to as "Compounds (II)"), from compounds one on the 13 /? - carbon atom (Ci- bis C.) alky! Substituted 8,14-Secogona-I, 3,5 (10), 9 (l 1), 15-pentaene-14,17-dione nucleus contain (hereinafter referred to as "compounds (I)"), using microorganisms.

Recently, methods for the total synthesis of 19-nor-steroids have been developed (see for example Miki and coworkers "Proceedings of the Chemical Society" 1963, p. 139; Crispin and coworkers, ibid, 1963, p 22, Windholz and co-workers "Journal of the Organic Chemistry". 28, page 1092, and Smith and coworkers »Experienta«, 19, page 394).

However, these processes have disadvantages when they are used for large-scale production: If a ver14-on-kern contains, with their enantiomers. To convert these compounds into natural oestrone and the like, they must be subjected to optical splitting at some suitable stage.

The subject matter of the invention is the process which can be carried out on an industrial scale and which is described in the patent claim for the preparation of the compounds (II) directly from the compound (I) in good yield.

The substituent on the 13 carbon atom is (Cr to Gt) -AIkylrest such as methyl, ethyl, propyl, butyl.

The compound (I) used as a starting material, which is produced, for example, by the method of NL-PS 66 12 205 (designed March 1, 1967) can be produced, carries a (Ci- to C4) -alkyl substituent on the 13-carbon atom and any 1 to 3 other substituents in the 1-, 2-, 3-, 4-, 6-, 7-, 11-, 12-, 15- or 16-position from the group consisting of oxygen groups, halogen groups and lower alkyl groups.

Examples of oxygen groups are hydroxyl groups and lower acyloxy groups with 1 to 4 carbon atoms and alkoxy radicals with 1 to 4 carbon atoms and, as halogen atoms, for example fluorine, Chlorine and bromine in question. As lower alkyl radicals, for example, straight-chain radicals with 1 to 4 carbon atoms or three-membered or six-membered rings including those mentioned above as substituents Residues that contain one or more oxygen groups in question.

Examples of starting compounds for the process according to the invention are:

8,14-Secoöstra-1,3,5 (10), 9 (11), 15-pen ta-

14,17-dione,
3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (11), 15-pen-

taen-14,17-dione,
3-ethoxy-8,14-secoöstra-1, 3,5 (10), 9 (11), 15-pen-

taen-14,17-dione,

S-methoxy-n-ethyl-e.H-secogonal, 3.5 (10), 9 (ll), 15-pentaen-14,17-dione,

3-ethoxy-13-ethyl-8,14-secogona-1,3,5 (10), 9 (11), 15-

pentaen-14,17-dione,
3-methoxy-13-isopropyl-8,14-secogona-

l, 3.5 (10), 9 (11) ₁ 15-pentaen-14,17-dione,
β--, 3-ethoxy-13-isopropy 1-8,14-secogona-

I, 3,5 (lü), 9 (ll), l5-pentaen-14, l 7-dione,
3-benzyloxy-8,14-secoöstra-l, 3,5 (10), 9 (ll), 15-pen-

taen-14, l7-dione.

2,3-dimethoxy-13-ethyl-8,14-secogona-

13 ^ (lO), 9 (n), l5-pentaen-l4, l7-dione,
S-methoxy-e-methyl-eM-secoöstra-

13,5 (10), 9 (11), 15-pentaen-14,17-dione and
a-Methoxy-ee-dimethyl-eH-secoöstra-U, 5 (10), 9 (II), 15-pentaene-14,17-dione.

The desired compounds (H) belong to the following Gn'j> pen:

IO

1) Compounds with one on the 13/3 carbon atom (Ci- to Ct) -alkyl-substituted 17 / J-hydroxy-8,14-secogona-U> 5 (10), 9 (l l), 15-pentaen-14-on-kernel produced by microorganisms such as Debaryomyces vini, Pichia wickerhamii, Pichia pijperi, Hansenula holstii and Hansenula capsulata are formed;

2) Compounds that have one on the 13/3 carbon atom (Ci- to Ct) -alkyl-substituted 17 «-hydroxy-8,14-

secogona-1,3,5 (10), 9 (11), 15-pentaen-14-on-kern
and are formed by microorganisms such as Kioeckera Msgna;

3) Compounds that have one on the 13 / J carbon atom (Ci- to QJ-alkyl-substituted 17 /? - hydroxy-8,14-secogona-1,3,5 (10), 9 (l l) -tetraen-14-on-kem included and by microorganisms such as Candida utilis, Debaryomyces nicotianae, Pichia etchellsii, Rhodotorula rubra, Schizosaccharomyces pombe, Hansenula saturnus, Hansenula holstii, formed will.

JO

The culture media for the growth of these microorganisms contain carbon and nitrogen sources, which are assimilable and usable by the microorganisms, and can include inorganic salts, various Contain vitamins and amino acids. js

Particularly suitable carbon sources are, for example, glucose, sucrose, dextrin, glycerin, etc. and all nitrogen sources, for example inorganic nitrogenous materials such as peptone, meat extract, Casein, corn steep liquor, dry yeast and yeast extract, and inorganic nitrogen compounds such as Ammonium nitrate, ammonium phosphate and ammonium sulfate.

Examples of the above-mentioned inorganic salts are sodium chloride, potassium sulfate and magnesium sulfate. The nutrients that favor the growth of the microorganisms are used in appropriate proportions used in a culture medium. The cultivation can arbitrarily as shaking culture, stationary Culture and submersculature with movement and ventilation take place.

The starting compound (I) can either be Start of cultivation or at a suitable phase in the course of cultivation continuously or at intervals or added all at once. The final concentration of the compound (I) in the medium is preferably about 0.05 to 0.6% (weight / volume).

The compound (I) can be in the form of powder or as a solution or suspension in an appropriate Solvents, e.g. B. acetone, methanol, ethanol, ω ethylene glycol, propylene glycol, dimethylformamide or Dioxane, with or without the addition of a surfactant, dispersant and the like can be used.

When the microorganism is used after being separated from the culture liquid, the bs Microbial cells in a buffer solution of suitable pH and suitable; - ionic strength or in water suspended and then brought together with the starting compound (I) to convert them.

The reaction proceeds usually at tinem pH of about 2 to 11 and oei a temperature between about 10 and 50 ° C, preferably between about 25 and 40 ⁰ C, for a period of about 2 days. However, the optimal conditions vary with factors such as the starting compounds and the microorganisms, and it is appropriate to select the optimal conditions in each individual case.

The end product (II) formed and enriched in this way in the reaction medium can according to various known methods are separated therefrom, e.g. B. by adsorption on a suitable Adsorbents, e.g. B. silica gel, and subsequent Elution with a suitable solvent, e.g. B. a benzene-acetone mixture or chloroform-acetone mixture, or by taking advantage of the difference in the partition coefficient between two liquid phases for example with countercurrent distribution or after customary chromatographic metals.

The optically active 17-hydroxy-8,14-secogona-pentaen-14-one nucleus which is substituted on the 13 carbon atom (Ci- to C4) -alkyl Compounds containing are converted into the corresponding tetraene using the enzyme system of a yeast, ζ B. Candida solani convicted.

The optically active compounds prepared in the manner described above which have one on the 13j3 carbon atom (Ci- to C4) -alkyl-substituted 17-hydroxy-e.H-secogona-tetraen-H-one nucleus are contained, for example, according to the method described in "Chemistry and Industry" 1966, p. 1340, converted to estrone and other 19-nor steroids.

Currently preferred embodiments of the invention are described in the following examples, in which the percentages are based on weight / volume. Parts by weight relate to parts of space how grams to cubic centimeters.

The microorganisms that were used in the experiments described in the examples are at Infinite for Fermentation, Osaka, Japan under the accession numbers which are used in the examples as "IFO numbers" are named, deposited. Some microorganisms that are identified by "N.R.R.L." with a number are on file with the Northern Regional Research Laboratory, USA

example 1

40 parts by volume of a liquid medium (pH 5.8), the 0.3% yeast extract, 0.5% polypepto-. Contains 0.2% corn steep liquor, 5% glucose and 5% sucrose, inoculated microorganisms mentioned with a seven-day slant culture of the vain \ p Table 1 below. The inoculated medium is kept at 28 ° C. for 2 days. After this time a solution of 0.0? Parts by weight

yOJOJ
14,17-dione (hereinafter referred to as "substrate (I)") was added in 0.8 parts by volume of ethanol. The cultivation is continued for another 2 days. The resulting broth is then mixed in two portions with a total of the same amount of ethyl acetate. The reaction products converted into the ethyl acetate phase are washed with water, dehydrated and concentrated to dryness.

Column chromatography is using carried out by silica gel. The reaction products are isolated from the fraction that comes with a 9: 1 mixture has been eluted by benzene and acetone. the

Products are determined according to their rotational dispersion curves in ethanol. Fi g. l (a) to (g) shows the Spectra of these compounds and other closely related steroids:

a) 3-methoxy-8.14secoöstra-l, 3,5 (10), 9 (II) -tetraen-170-ol-14-one (designated in the table as β3 );

b) 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (1l), 15-pentaen-170-ol-14-one (designated in the table as Δ "-ββ );

c) 3-methoxy-13 «-methyl-8,14-secogona-1,3,5 (10), 9 ( 11) -tetraen-17 /? -Ol-l 4-one;

d) 3-methoxy-13i \ -methyl-8,14-secogona-1,3.5 (10), 9 (II), 15-pentaen-17j3-ol-14-one;

c) Mixture of S-methoxy-SH-sccoöstra-1.3,5 (10), 9 (] l) -tetraen-l7 / 3-ol-l4-one and
3-methoxy-8,14-secoestra-I, 3.5 (10), 9 (11) .15-pentaen-! 7/3-ol-14-one;

f) mixture of 3-methoxy-13.x-methyl-8,14-secogona-1,3.5 (IO), 9 (l l) -tetraen-l7 / J-ol-l4-on and

3-methoxy-13 "-methyl-8,) 4-secogona-1,3,5 (10), 9,1 5-pentaen-170-ol-14-one and
g) 3-MeIhOXy-S ₁ M-SeCOOsIrB-1,3,5 (10), 9 (11), 15-pentaen-17λ-οΙ-1 4-one (referred to in the table as Δ ^ -stx ) .

The samples obtained from FIG. 1 cannot be distinguished from one another, e.g. B. 3-methoxy-8,14-seco östra-1,3,5 (10), 9 (U), 15-pentaen-170-ol-14-one um

3-methoxy-13 "-methyl-8,14-secogona-1,3,5 (10), 9 (11), 15 pentaen-l7 ^ -ol-14-one, be in methanol together with a small amount of p-toluenesulfonic acid Left to stand room temperature and on the formation of 3-methoxy-9,14-oxido-8,14-secoöstra-1,3,5 (10), 15 tetra ^ n - ^ - on by thin layer chromatography un investigated (a 9.14 oxide only occurs in the case of the latter called connection). In this way it is determined whether the steric orientation / wipe dei I3-methyl group and the 17-hydroxyl group eis odei is trans.

Microorganism IFO no. product sales NRRL no. Wt% Candida utilis (1086) ßß 72 Debaryomyces nicotianae (0855) ßß 75 Debaryomyces vini (1214) A ^ -ßß 68 Debaryomyces vanriji (1285) "-ßß 70 Kloeckera magna (0868) / 115-00 75 Piöhia wickerhamii (1278) Δ ^ -ββ 65 Pichia etchellsii (1283) ßß 70 Pichia pijperi (1290) Δ "-ββ 80 Rhodotorula rubra (0891) ßß 75 Schizosaccharomyces pombe (0362) ßß 78 Hansenula holstii (0980) ßßA »-ßß 78 NRRL-Y-2155 Hansenula capsulata (0984) Δΐί-ββ 70 NRR1-Y-1842 Hansenula saturnus (0993) ßß 75

Example 2

snmiAil. » pinAc fli'iccicron MpHinmQ inH SRJ Hac

0.3% yeast extract, 0.5% proteose peptone (Difco), 0.2% corn steep liquor, 5% glucose and 5% sucrose are inoculated with a culture of Debaryomyces nicotianae IFO-0855. The microorganism is ^{grown at 28 °} C. for 2 days. Then a solution of 1 part of the substrate (1) in 40 parts by volume of ethanol is added to the culture, which is then incubated for a further 2 days. The reaction product is extracted twice with 1000 parts by volume of ethyl acetate and, after the solvent has been distilled off, purified by column chromatography on silica gel with a benzene-acetone mixture as the eluent. The solvent is distilled off from the fraction containing the product. The residue is recrystallized from ethanol, 0.42 parts by weight of 3-methoxy-8,14-secoöstra-13.5 (10), 9 (l I) -tetraen-17β-ol-14-one as colorless platelets with a melting point of 112-113 ^s C can be obtained. The compound shows the negative Cottone effect curve and a specific rotation [λ] »of -39 ° (C = 03% in dioxane).

Example 3

In the manner described in Example 2, a solution of 1 part by weight! of the substrate (I) in 40 parts by volume Ethanol to 1000 parts by volume of a two-day culture

given by Pichia etchellsii (IFO-1283), which is then incubated for two more days. The reaction product is pYtrahiprt Hiirrh ^ äiilpnrhrnmatn with Äthvlarptat.

purified graphie and left to crystallize from ethanol, with 0.52 parts by weight of 3-methoxy-8,14-secoöstra-1,3,5 (10) .9 (II) -tetraen-170-ol-14-one as colorless platelets with a melting point of 111-113 ° C.
[*]: - · -39 ° (C =], ^{0 0} Zo dioxane).

Example 4

In the manner described in Example 2, 8 parts by volume of a 2.5% strength solution of the substrate (I) wind in Ethanol given to 200 parts by volume of a two-day culture of Hansenula holstii (IFO-0980), which is then added to 28 ° C is incubated Samples are taken 12, 24, 36 and 48 hours after the addition of the substrate (I). The samples are each extracted with ethyl acetate Extracts are purified by column chromatography on silica gel and dissolved in ethanol

The rotation dispersion of the ethanolic solution is measured. The results are shown in Figure 2 (a, b, c and d).

These rotational dispersion curves vary with the Cultivation (reaction) time. For example, the curves are correct for the products that have a cultivation period of 12, 24, 36 and 48 hours correspond to the curves for S-methoxy-e.M-secoöstra-

l3,5 (10), 9 (1l), 15-pentaen-17 /? - ol-14-one, a mixture of 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (II), 15-pentaen-17j3-ol-14-one and S-methoxy-e.U-secoöstra-I, 3,5 (10), 9 (11) -tetraen-17jj-ol-l4-one or 3-methoxy-8,14-secoöstra-1,3,5 {10), 9 ( 11) -tetraen-170-ol-14-one.

While the product, which has the reaction time of 1? Hours corresponds, not crystallized, shows its in a chloroform solution recorded absorption spectrum the absorption for hydroxy (3620 cm- ') and «, / ^ - unsaturated ketone (1680 cm ~ ').

The rotary dispersion of this product shown in FIG. 2a gives the negative Cottone effect curve with one foot at 360 Γημ. Since neither 3-methoxy-9,14-oxido-8,14-secoöstra-1,3,5 ( 10), 9 (11) -pentaen-17-one nor its enantiomer is formed when this product is treated with a small amount of p-toluenesulfonic acid is treated in benzene, it is evident that this product is the compound S-methoxy-e.H-secoöstra-1,3,5 (10), 9 (11) .15-pentaen-17/9-ol-14-one is.

Which is obtained after a culture period of 36 or 48 hours, the product is, from ethanol in the form of colorless flakes of melting point 111-113 ⁰ C crystallized (yield 78 wt ° / o). The specific rotation [λ] '"' and the infrared spectra of the products agree completely with those of S-methoxy-eH-secoöstra-1,3,5 (10), 9 (II) -tetraen-170-ol-14-one match.

From the above it can be concluded that Hansenula holstii IFO-0980 has the ability to first remove the 17-carbonyl group of the substrate (I) with the formation of 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (ll) to reduce 15-pentaen-17j3-ol-14-one and then the zl ^{15 of} the latter to 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (l I) - tetraen-17 /? - ol-14-one to reduce.

Example 5

In the manner described in Example 2, a solution of 0.5 part by weight of the substrate (I) in 40 Volume parts of ethanol given to 1000 volume parts of a two-day culture of Debaryomyces vanriji IFO-1285, which is then cultivated for 2 days. The reaction product is extracted with ethyl acetate and the extract purified by column chromatography in the manner described in Example 2, 0.35 part of 3-meth-

ygU.f ^
14-one can be obtained as an oil.

The rotary dispersion of the in ethanol solution

κι dissolved oily product is identical to that in Fig. L (b) shown rotational dispersion. If the product is treated with p-toluenesulfonic acid, it occurs no 9,14 oxide.

'"' Exampleö

In the manner described in Example 2, 40 parts by volume of a 2.5% strength solution of the substrate (I) are added Ethanol to 1000 parts by volume of a two-day culture of

_'ii Kloeckera magna IFO-0868 and then leaves the reaction Allow 2 days at 28 ° C. After this time the product is extracted with ethyl acetate.

The extract is purified by column chromatography on silica gel in the manner described in Example 2

r, purifies, whereby 3-methoxy-8,14-secoöstra-1,3,5 (10), 9,15-pentaen-17 <x-ol-14-one is obtained as an oil (yield 75% by weight).

The nuclear magnetic resonance spectrum of the product in CDCh (D; deuterium) shows peaks which are relevant for the

jo /) 'M7-ol structure: 17-H (r 5.52, doublet), 16-H (τ 2.62. Quartet) and 15-H (r 3.89 quartet) are characteristic.

The rotational dispersion of the product, measured in an ethanol solution, gives the shown in Fig. 1 (g)

j) showed positive cotto effect curves. The first peak occurs at 360 ιτιμ. This product results from treatment with p-toluenesulfonic acid in benzene a 9,14-oxide.

For this purpose 3 sheets of drawings

Claims (1)

Claim: Process for the preparation of optically active steroid compounds that have one on the 130 carbon atom (Cr to Ct) -alkyl-substituted 17-hydroxy-8,14-secogonapolyen-14-one nucleus contain, characterized in that one