HK1081999B - Microbiological method for the production of 7 alpha-substituted 11 alpha-hydrox-ysteroids - Google Patents

Description

Microbial process for the preparation of 7 alpha-substituted 11 alpha-hydroxy steroids

Technical Field

The invention relates to a microbiological method for producing 7 alpha-substituted 11 alpha-hydroxy steroids and to 7 alpha, 17 alpha-substituted 11 beta-halogen steroids which can be produced therefrom, to a method for producing the latter, to the use thereof and to pharmaceutical preparations containing said compounds. Furthermore, the present invention relates to other 7 α -substituted 11 β -halo steroids, i.e. 7 α -substituted estra-1, 3, 5(10) -trienes obtainable from 7 α -substituted 11 α -hydroxy steroids.

Background

Androgens, particularly testosterone, are used to treat andropause symptoms and for male sexual organ development and male birth control. In addition, these hormones also have partial anabolic active ingredients which can promote, for example, muscle growth.

Andropause is characterized by a decrease in age-related endogenous androgen production and is therefore treated with hormone replacement therapy (HRT: hormone replacement therapy).

In addition to the reduction in spermatogenesis, administration of LH-RH for Male birth control also results in the release of LH and a decrease in Testosterone levels and libido which are counteracted by the administration of Testosterone drugs (D.E. Cummings et al, "State-releasing Effects of the content antagonist 7 α -Methyl-19-Testosterone: A content Alternative to Testosterone for the nutrient Replacement and Male content" Journal of clinical Endocrinology and Metabolism, Vol.83, No.12, pages 4212-.

Combination therapy with androgen and progestin active ingredients can be used to control male fertility (see, e.g., WO 01/60376 a and references cited therein).

In the case of treatment with testosterone, it was found that side effects develop in particular in prostate hypertrophy (BPH: benign prostatic hyperplasia) due to an increased number of stromal cells and glands. In the 5 alpha-reductase regulated metabolism of progesterone, Dihydroprogesterone (DHT) is formed, which in turn can lead to the development of, for example, BPH (Cummings et al, supra; WO 99/13883A 1). Thus, BPH (finasteride) is treated in clinical practice by inhibition of the 5 α -reductase.

The rapid metabolism of the androgenic steroid testosterone in the human body further not only leads to the harmful production of DHT, but also necessitates the oral administration of higher doses to achieve the desired therapeutic level of testosterone. Therefore, other alternative formulations such as intramuscular injection or large patch (largepatch) must be used.

In order to replace testosterone in the field of the above indications, 7 α -methyl-19-nortestosterone (MenT) has been proposed, which on the one hand has A higher biological effect than testosterone due to A higher binding to the androgen receptor and on the other hand may hinder the metabolism of the 5 α -reductase due to steric hindrance of the 7 α -methyl group (Cummings et al, suprA, WO 99/13883A 1, WO 99/13812A 1, US-A-5,342,834).

During testosterone metabolism, particularly in the brain, liver and adipose tissue, a small fraction of this compound also produces estradiol by aromatisation of the a ring of the steroid system. Estradiol is the essential cause of sex-specific behavior and gonadotropin regulation in terms of the overall function of testosterone and its metabolites. Thus, its function is considered beneficial just as testosterone does in adult males (Cummings et al, supra).

However, the pharmacokinetics of testosterone have been found to be unsatisfactory. In particular, upon oral administration, testosterone is rapidly excreted, making the effectiveness and duration of action of the medicaments prepared therefrom less than satisfactory. Thus other testosterone derivatives were synthesized. Such derivatives are described, for example, in US-A-5,952,319, in particular the 7 α, 11 β -dimethyl derivatives of 19 nortestosterone, namely 7 α, 11 β -dimethyl-17 β -hydroxyestr-4-en-3-one, 7 α, 11 β -dimethyl-17 β -heptyloxyestr-4-en-3-one, 7 α, 11 β -dimethyl-17 β - [ [ (2-cyclopentylethyl) -carbonyl ] -oxy ] -estr-4-en-3-one, 7 α, 11 β -dimethyl-17 β - (phenylacetyloxy) -estr-4-en-3-one and 7 α, 11 β -dimethyl-17 β - [ [ (trans-4- [ n-butyl ] cyclohexyl) -carbonyl ] - Oxy ] -estr-4-en-3-one.

The aforementioned 7 α, 11 β -dimethyl derivatives, such as MeNT, have the aforementioned advantages, including improved pharmacokinetic properties, e.g. improved efficacy and duration of action compared to testosterone. However, these derivatives can only be prepared by expensive synthetic methods.

Microbial methods for the synthesis of steroids are described in EP 0900283B 1. It is indicated that estr-4-ene-3, 17-dione and canrenone can be converted into the corresponding 11 α -hydroxy analogue using a microorganism selected from the group consisting of: aspergillus niger (Aspergillus niger), Rhizopus arrhizus (Rhizopus arrhizus) and Pestelotia strains. However, Shibahara et al, Biochim. Biophys. acta, 202(1970), 172-179 are also referred to in the description, which reports that microbial 11 α -hydroxylation in steroids may not be predictable.

The problem underlying the present invention was therefore to find testosterone derivatives which are insensitive to 5 α -reductase, have improved pharmacokinetic properties and are particularly easy to prepare. Thus, a very interesting aspect of the present invention consists in finding a process in which the initial product is easy to produce.

The problem on which the invention is based is solved by: a process for the microbiological preparation of 7 α -substituted steroids as claimed in claims 1, 3 and 6 and 7 α, 17 α -substituted 11 β -halo steroids as claimed in claim 11, a process for the preparation of 7 α, 17 α -substituted 11 β -halo steroids as claimed in claims 23, 24 and 25, the use of 7 α, 17 α -substituted 11 β -halo steroids as claimed in claim 26, pharmaceutical formulations containing these 7 α, 17 α -substituted 11 β -halo steroids as claimed in claim 27 and 7 α -substituted 11 β -haloestra-1, 3, 5(10) -trienes as claimed in claim 21. Preferred embodiments of the claimed subject matter are set forth in the dependent claims.

Definition of

The following definitions relate to the various parts of the specification and claims and the attached drawing I:

all radicals, groups or other structural units can in each case be varied independently of one another within the meaning indicated.

All alkyl, alkylene, alkenyl, alkenylene, alkynyl and alkynylene groups may be straight-chain or branched. For example, the propenyl group may be represented by one of the following chemical structures: -CH ═ C-CH₃、-CH₂-C＝CH₂or-C (CH)₃)＝CH₂. Thus, for example, methyl, ethyl, n-propyl, isopropyl-n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 1-methyl-n-pentyl, 2-methyl-n-pentyl, 3-methyl-n-pentyl, 4-methyl-n-pentyl, 1-ethyl-n-butyl, 2-ethyl-n-butyl and the like all belong to the group C₁-C₁₈An alkyl group.

Cycloaliphatic alkyl is cycloalkyl or is cycloalkyl substituted by one or several alkyl groups, which are bonded directly through the cycloalkyl ring or through one of the alkyl groups.

Likewise, an alicyclic alkenyl group is a cycloalkenyl group or a cycloalkenyl or cycloalkyl group substituted by one or more alkenyl groups or one or more alkyl groups, bonded directly through the cycloalkenyl ring or through one of the alkenyl or optional alkyl groups, wherein the alicyclic alkenyl group contains at least one double bond.

In another aspect, the aryl group can be phenyl, as well as 1-naphthyl or 2-naphthyl. In principle, aryl also includes heteroaryl, in particular 2-, 3-and 4-pyridyl, 2-and 3-furyl, 2-and 3-thienyl, 2-and 3-pyrrolyl, 2-, 4-and 5-imidazolyl, pyridazinyl, 2-, 4-and 5-pyrimidinyl and 3-and 4-pyridazinyl.

Halogen is fluorine, chlorine, bromine or iodine.

Pharmaceutically compatible addition salts are the salts of the corresponding compounds with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, citric acid, oxalic acid, tartaric acid and methanesulfonic acid. Especially with succinic acid.

The number of superscripts being R, e.g. R¹³Refers to its position on the steroid ring backbone, the carbon atoms on the steroid ring backbone being numbered according to IUPAC nomenclature. Superscripts on the marks C, e.g. C¹⁰Refers to the position of each carbon atom in the steroid ring backbone.

Disclosure of Invention

The preparation of 7 α -substituted 11 α -hydroxy steroids of general formula 4, B using a novel microbial process:

wherein

R⁷Is a P-Q group, and

p is C₁-C₄Alkylene, Q is hydrogen, C₁-C₄Alkyl or C₁-C₄Fluoroalkyl (partially or fully fluorinated alkyl), and the P-Q group is bonded to the steroid skeleton via P,

R¹⁰representative H, CH₃Or CF₃And an

R¹³Is methyl or ethyl.

In a first method embodiment for the preparation of these substances, a suitable 7 α -substituted steroid of the general formula 3, a is hydroxylated and oxidized in one process step:

wherein R is⁷、R¹⁰And R¹³Having the same meaning as described in general formula 4, B, said hydroxylation and oxidation using a microorganism selected from the group consisting of: aspergillus (Aspergillus sp.), Beauveria bassiana (Beauveria sp.), and Pleurotus plexis (Glomer)ella sp.), Japanese shell (Gnomonia sp.), megamonospora (Haplosporilla sp.), and Rhizopus (Rhizopus sp.). Particularly preferred are Aspergillus awamori (Aspergillus awamori), Aspergillus ficorum (Aspergillus fischeri), Aspergillus syphilis (Aspergillus malignaus), Aspergillus niger (Aspergillus niger), Beauveria bassiana (Beauveria bassiana), Rhizopus malorum (Glomerella cingulata), Gnomonicacina, Haplosporiella heperiana and Rhizopus stolonifer (Rhizopus stolonifer), with Aspergillus awamori (CBS), Aspergillus ficuus (ATCC1020), Aspergillus syphilis (IMI 16061), Aspergillus niger (ATCC 9142), Bombyx mori (ATCC 7159), Rhizopus malorum mali (CBS15226, CBS 23849, CBS 98069, ATCC 56596, ATCC 64682, IFO 6425), Aspergillus fumigatus 15226, Rhizopus reticulata (CBS 15215226), Rhizopus pustus 20841 and Gluconobacter stolonifera (CBS 20841).

Alternatively, the microbiological process can be carried out in two steps, wherein the hydroxylation and oxidation reactions take place in successive reaction steps. The reaction process can be controlled by the reaction time: by interrupting the reaction, for example after a certain reaction time, hydroxylated but still unoxidized species can be obtained. Thus, the two process steps can be carried out separately or mixed.

To this end, the compound of formula 3, a may be hydroxylated at the 11-position in a first microbiological process step using a first microorganism selected from the group consisting of: aspergillus (Aspergillus sp.), Beauveria (Beauveria sp.), Gibberella (Gibberella sp.), Glomeella sp., Japanese (Gnomonia sp.), Metarhizium (Metarhizium sp.), Oryctolagi (Nigrospora sp.), Rhizopus (Rhizopus sp.), and Verticillium (Verticillium sp.) produce 7 α -substituted steroids having a hydroxyl group at the 11 α -position. The compounds have the general formula C:

wherein R is⁷、R¹⁰And R¹³Has the same meaning as that described in the general formula 4, B. Specifically used are Aspergillus toxicidus (Aspergillus malignaus), Aspergillus melleus (Aspergillus melleus), Aspergillus niger (Aspergillus niger), Aspergillus ochraceus (Aspergillus ochraceus), Beauveria bassiana (Beauveria bassiana), Rhizopus oryzae (Gibberella fujikuroi), Gibberella zeae (Gibberella zeae), Rhizoctonia malus (Glomelas) cinerea), Glomelas fumarides, Gomonia cingulata, Metarhizium anisopliae (Metarhizium anisopliae), Nigrospora sphaerica (Nigrospora sphaerica), Rhizopus oryzae (Rhizopus oryzae), Rhizopus stolonifer (Rhizopus stolonifer) and Verticillium (Verticillium dahlia). Particularly preferred for hydroxylation in this connection are Aspergillus fumigatus (IMI 16061), Aspergillus melleus (CBS), Aspergillus niger (ATCC 11394), Aspergillus ochraceus (NRRL 405, CBS 13252, ATCC 46504), Beauveria bassiana (ATCC 7159, IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS12736), Gibberella oryzae (ATCC 14842), Gibberella zeae (CBS 4474), Rhizoctonia mali (ATCC 10534, CBS 23849, CBS 23749, ATCC 16646, ATCC 16052, IFO6459, IFO 6470, CBS 98069, IFO 7478, IFO 5257, ATCC 64682, ATCC 70), Glomella fuscarideaides (ATCC 9552), Glomella cingulata (CBS15226), Metarrhizium anisopliae (IFO 5940), Rhizopus nigricans (ATCC 34172), ATCC 15434158, ATCC 15458), Mycoplasma sphaeroides (ATCC 34141), and Mycoplasma sphaeroides (ATCC 34141).

The intermediate product C is subsequently oxidized in a second microbiological process step by using a second microorganism selected from the group consisting of: bacillus (Bacillus sp.), Mycobacterium (Mycobacterium sp.), Nocardia (Nocardia sp.), and Pseudomonas (Pseudomonas sp.). Specifically used are Bacillus lacticus, Bacillus sphaericus, Mycobacterium neoaurum, Mycobacterium smegmatis, Nocardia corallina, Nocardia globerula, Nocardia mimicus, Nocardia restricta, Nocardia rubra, Nocardia salmoniliforme and Pseudomonas testosteroni. Among them, particularly preferably used are Bacillus lactis (ATCC 245), Bacillus sphaericus (ATCC 7055), Mycobacterium neoaurum (ATCC 9626, NRRLB-3683, NRRL B-3805), Mycobacterium smegmatis (ATCC 14468), Nocardia corallina (ATCC 31338), Nocardia globisporus (ATCC 9356), Nocardia mimicus (ATCC 19150), Nocardia restricta (NCIB 10027), Nocardia rubra (ATCC 14352), Nocardia salmon (ATCC 19149) and Pseudomonastes testosterone (ATCC 11996).

In a second process embodiment, the compounds of formula 4, B can be prepared in a microbiological reaction from 7 α -substituted steroids of formula D:

wherein R is⁷、R¹⁰And R¹³Have the same meanings as described for the compounds of the general formula 4, B. The reaction is carried out using a microorganism selected from the group consisting of: aspergillus (Aspergillus sp.), Beauveria (Beauverian sp.), Curvularia (Curvularia sp.), Gibberella (Gibberella sp.), plexiglas (Glomeellasp.), Uvaria (Gnomonia sp.), Gymnocladium (Gnomonia sp.), Monospora magna (Haplosporilla sp.), Torulopsis circinella (Helicostylum sp.), Blastomyces Nigrospora (Nigrospora sp.), Rhizopus (Rhizopus sp.), and Rhizopus comycoris (Syncephalastrum sp.). Wherein the 11 α -position of the steroid skeleton is hydroxylated to form a 7 α -substituted 11 α -hydroxy steroid of formula 4, B. Preferred are Aspergillus oniae (Aspergillus alliacea), Aspergillus awamori (Aspergillus awamori), Aspergillus fischeri (Aspergillus fischeri), Aspergillus toxicus (Aspergillus malignaus), Aspergillus melleus (Aspergillus mellus), Aspergillus nidulans (Aspergillus nidulans), Aspergillus niger (Aspergillus niger), Aspergillus ochraceus (Aspergillus ochraceus), Aspergillus versicolor (Aspergillus variegatus), Beauveria bassiana (Beauveria) and Aspergillus awamoriBasisana), Curvularia lunata (Curvularia lunata), Gibberella zeae (Gibberella zeae), Rhizopus mali (Glomerella cingulata), Glomeella fuscarinates, Gnomonia cingulata, Haplosporella heperiana, Gilles pyriformis (Helicostylum piriformis), Nigrospora sphaericus (Nigrospora sphaerica), Rhizopus oryzae (Rhizopus oryzae) and Rhizophora racemosa (Syncephalatum racemosus), of which Aspergillus cepacia (ATCC10060), Aspergillus awamori (CBS), Aspergillus Philliae (ATCC1020), Aspergillus toxicorusflavus (IMI 16061), Aspergillus melleus (CBS), Aspergillus nidulans (ATCC 11267), Aspergillus niger (ATCC 9142, ATCC11394), Aspergillus ochraceus (NRZ) and ATCC 6783, ATCC 4826, ATCC 4874, ATCC 368454, Aspergillus oryzae (ATCC 3644, ATCC 36363644, ATCC 365854, Aspergillus kayami (ATCC 3635, ATCC 363635, ATCC 368474, ATCC 3635, ATCC 368454, ATCC 3635, CBS 23749, ATCC 16646, IFO6459, IFO 6425, IFO 6470, ATCC 15093, ATCC 10529, IFO 5257, ATCC 56596, ATCC 64682), Glomeella fuscarides (ATCC 9552), Gnomonia cingulata (CBS15226), Haplosporella heperidica (CBS 20837), Torulopsis piricola (ATCC 8992), Blastomyces sphaericus (ATCC 12772), Rhizopus oryzae (ATCC 4858), and Conidiobolus racemosus (IFO 4827).

Particularly suitable are processes for the formation of 7 α -substituted 11 α -hydroxy steroids of the general formula 4, B, wherein R independently of one another⁷Represents CH₃And/or R¹⁰Represents H and/or R¹³Represents CH₃。

The method is carried out in a conventional manner. For this purpose, usually, a sterilization nutrient solution is first prepared for the strain, and then the nutrient solution is inoculated with a culture solution of the strain to culture the strain. The preculture thus prepared is then added to a fermenter which may optionally be covered with a suitable nutrient solution. The starting substances, in this connection the compounds of the formula 3, A or the compounds of the formula D, are preferably added to the fermenter after the growth phase of the strain culture, so that the reaction according to the invention can be carried out. After the reaction is complete, the mixture is purified and isolated by conventional methods to provide the desired 7 α -substituted 11 α -hydroxy steroid.

From the thus obtained compounds of the general formula 4, B, further compounds of the present invention can be synthesized by the production process of the present invention. In particular 7 α, 17 α -substituted 11 β -halogenated steroids of general formula 8, 10, 12 and their pharmaceutically compatible addition salts, esters, amides, form excellent active ingredients:

wherein

U-V-W-X-Y-Z represents one of the following ring structures: c¹-C²-C³-C⁴＝C⁵-C¹⁰、C¹-C²-C³-C⁴-C⁵＝C¹⁰、C¹-C²-C³-C⁴-C⁵-C¹⁰And in this case, an oxo group (═ O) is bonded to W (═ C)³) Or represents a ring structure C¹＝C²-C³＝C⁴-C⁵＝C¹⁰And in this case OR³Is bonded to W (═ C)³) In the above-mentioned manner,

R³representative H, C₁-C₄Alkyl radical, C₁-C₄Alkanoyl OR having OR³C of O atom of radicals₃-C₇A cyclic ether having a cyclic structure represented by the general formula,

R⁷is a P-Q group, and P is C₁-C₄Alkylene, Q is hydrogen, C₁-C₄Alkyl or C₁-C₄Fluoroalkyl (partially or fully fluorinated alkyl), and the P-Q group is bonded to the steroid skeleton via P,

R¹⁰may be in the alpha-or beta-position, which represents H, CH₃Or CF₃And is not C only in X-Y-Z⁴-C⁵＝C¹⁰When the pressure difference exists,

R¹¹is a halogen, and the halogen is a halogen,

R¹³is a methyl group or an ethyl group,

R¹⁷representative H, C₁-C₁₈Alkyl radical, C₁-C₁₈Alicyclic alkyl radical, C₁-C₁₈Alkenyl radical, C₁-C₁₈Alicyclic alkenyl radical, C₁-C₁₈Alkynyl, C₁-C₁₈Alkylaryl group, C₁-C₈Alkylene nitriles or represents a P-Q group, where the P-Q group has the abovementioned meaning,

R^17’representative H, C₁-C₁₈Alkyl radical, C₁-C₁₈Alicyclic alkyl radical, C₁-C₁₈Alkenyl radical, C₁-C₁₈Alicyclic alkenyl radical, C₁-C₁₈Alkynyl, C₁-C₁₈Alkylaryl group, wherein R^17’May also be bonded to 17 beta-oxy via a keto group, and R^17’May additionally be substituted by one or more NR¹⁸R¹⁹Radicals or one or more SO_xR²⁰Wherein x is 0, 1 or 2, and R is¹⁸、R¹⁹And R²⁰In each case independently of one another with R¹⁷The meaning of (a).

These compounds are obtainable by 7 α -substituted 11 α -hydroxy steroids of the general formula 4, B from an additional process step, which are valuable active ingredients with a strong androgenic action without the side effects mentioned above. These compounds are suitable for the production of medicaments, in particular potent contraceptive agents and active ingredients for Hormone Replacement Therapy (HRT).

Provided that R is^17’In addition to NR¹⁸R¹⁹And (b) a substituent which may be methylamino, dimethylamino, ethylamino, diethylamino, cyclohexylamino, dicyclohexylamino, phenylamino, diphenylamino, benzylamino, or dibenzylamino.

Particularly suitable 7 α, 17 α -substituted 11 β -halo steroids having the general formulae 8, 10, 12 are those wherein U-V-W-X-Y-Z represents the ring structure C¹-C²-C³-C⁴＝C⁵-C¹⁰、C¹-C²-C³-C⁴-C⁵＝C¹⁰Or C¹＝C²-C³＝C⁴-C⁵＝C¹⁰The compound of (1).

In the first case (U-V-W-X-Y-Z represents C¹-C²-C³-C⁴＝C⁵-C¹⁰) A steroid compound of general formula 10:

in the second case (U-V-W-X-Y-Z represents C¹-C²-C³-C⁴-C⁵＝C¹⁰) A steroid compound of formula 12:

the compounds of formulae 10 and 12 are androgen compounds.

In the third case (U-V-W-X-Y-Z represents C¹＝C²-C³＝C⁴-C⁵＝C¹⁰) A steroid compound of formula 8:

these compounds are estrogens (estrogen receptor binding compounds).

In the above three cases, R³、R⁷、R¹⁰、R¹¹、R¹³、R¹⁷And R^17’Have the same meanings as the corresponding radicals in the formulae 8, 10, 12.

Independently of one another, preferably R¹Represents H and/or R⁷Represents CH₃And/or R¹¹Represents fluorine and/or R¹³Represents CH₃And/or R¹⁷Representative H, CH₃Especially of ethynyl group₁-C₁₈Alkynyl, CH₂CN or CF₃And/or R^17’Represents H.

Among the 7 α, 17 α -substituted 11 β -halo steroids of formula 8, 10, 12 are particularly suitable for the present invention:

17 α -ethynyl-11 β -fluoro-17 β -hydroxy-7 α -methylestr-4-en-3-one (formula 10)

17 α -ethynyl-11 β -fluoro-17 β -hydroxy-7 α -methylestra-5 (10) -en-3-one (formula 12)

17 α -ethynyl-11 β -fluoro-7 α -methylestra-1, 3, 5(10) -triene-3, 17 β -diol (formula 8)

These compounds can be prepared by the following methods:

for the preparation of compounds in which U-V-W-X-Y-Z represents the ring structure C¹-C²-C³-C⁴＝C⁵-C¹⁰The 7 α, 17 α -substituted 11 β -halo steroid having the general formula 10, using the 7 α -substituted 11 α -hydroxy steroid having the general formula 4, B obtained by the microbiological preparation method of the present invention as a starting material.

In a first synthesis step, these thus obtained 7 α -substituted 11 α -hydroxy steroids are converted into the corresponding 7 α -substituted 11 β -halogen steroids 5 by nucleophilic substitution using a halodehydroxylating reagent:

all compounds which are customarily used for this purpose can be used as halogen dehydroxylating agents, for example hydrofluoric, hydrochloric, hydrobromic or hydroiodic acid, thionyl chloride or thionyl bromide, phosphorus pentachloride, phosphorus oxychloride, N-chlorosuccinimide, triphenylphosphine/carbon tetrachloride, HF/pyridine or diethylaminosulfur trifluoride or preferably nonafluorobutanesulfonyl fluoride (nonafluorofluoride)/1, 5-diazabicyclo [5.4.0] undecene.

Then through the ring skeleton C¹⁷Selective alkylation of (2) compound 10 was prepared from compound 5 (see figure 1). Common alkylating agents can be used for selective alkylation, such as Grignard compounds and organometallic compounds, especially alkyllithium compounds. For example, ethynylmagnesium bromide may be used as an alkylating agent to prepare the corresponding 17 α -ethynyl-17 β -hydroxyestr-4-en-3-one from estr-4-ene-3, 17-dione.

Compounds of formula 10 can be used and isomerized to delta⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾Double bonds, thereby being useful for the preparation of compounds in which U-V-W-X-Y-Z represents the ring structure C¹-C²-C³-C⁴-C⁵＝C¹⁰A 7 α, 17 α -substituted 11 β -halo steroid having the general formula 12. To protect the 3-keto group, a cyclic ether is first formed for this purpose in the 3-position. Then, a⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾A double bond to produce a 7 α, 17 α -substituted 11 β -halo steroid having the general formula 12, and the protecting group is cleaved again.

For the preparation of further compounds in which U-V-W-X-Y-Z represents a ring structure C¹＝C²-C³＝C⁴-C⁵＝C¹⁰The 7 α, 17 α -substituted 11 β -halogen steroid compound having the general formula 8, which process is as follows:

first, the corresponding 11 β -halo steroids of formula 5 are prepared using the 7 α -substituted 11 α -hydroxy steroids of formula 4, B prepared by microbiological hydroxylation and oxidation by halodehydroxylation in a nucleophilic substitution reaction as described above.

From the former, by oxidation, for example, with copper (II) salts, 7 α -substituted estra-1, 3, 5(10) -trienes of the general formula 6 are formed:

wherein R is³、R⁷、R¹¹And R¹³The meaning of (A) is as defined above. If R is³Representing H, these compounds can be directly synthesized. If another group replaces R³H in (b), the corresponding ether or ester must be prepared by known methods after oxidation to form the 1, 3, 5(10) -triene ring.

Since the 7 α -substituted 11 β -halogen estra-1, 3, 5(10) -trienes of formula 6 and their pharmaceutically compatible addition salts, esters and amides are novel, protection is required in the present invention as intermediates for the synthesis of 7 α, 17 α -substituted 11 β -halogen steroids of formula 8.

A particularly preferred 7 α -substituted 11 β -halogen estra-1, 3, 5(10) -triene of the formula 6 is 11 β -fluoro-3-hydroxy-7 α -methylestra-1, 3, 5(10) -trien-17-one.

The 7 α, 17 α -substituted 11 β -halogen steroids of the general formula 8 according to the invention can be prepared from the 7 α -substituted 11 β -halogen estra-1, 3, 5(10) -trienes of the general formula 6, by a process which is as described above by selective oxidation of the ring skeleton C¹⁷The method for preparing formula 10 is the same.

Furthermore, 7 α -substituted 11 β -halo steroids of formula 9 can also be prepared by substances of formula 4, B prepared by microbiological hydroxylation and oxidation of formula 3, a or D, and said 7 α -substituted 11 β -halo steroids also have an androgenic effect:

wherein R is⁷、R¹¹And R¹³The meaning of (A) is as defined above. One particularly preferred compound is 11 β -fluoro-17 β -hydroxy-7 α -methylestra-4-en-3-one. The compounds of formula 9 and their pharmaceutically compatible addition salts, esters and amides also have androgenic effects.

To prepare the compounds of general formula 9, estra-4-ene-3, 17-dione 5 is reduced with boron hydride to 17 β -hydroxy-estra-4-ene-3-one 9.

In addition, compounds of formula 9 can be converted to the corresponding 7 α -substituted 11 β -halo estr-5 (10) -enes:

wherein R is⁷、R¹⁰、R¹¹And R¹³The meanings of (A) are as described in the general formulae 8, 10, 12. To this end, Δ of the compound of the formula 9⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾A double bond. To protect the 3-keto group, a cyclic ether is first formed for this purpose in the 3-position. Then, a⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾Double bond to produce a 7 α -substituted 11 β -halo steroid and the protecting group is cleaved again.

Finally, the corresponding 7 α -substituted 11 β -haloestr-5 (10) -enes are obtained by reacting Δ⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾The double bond is converted from a compound of formula 5.

Wherein R is⁷、R¹⁰、R¹¹And R¹³The meanings of (A) are as described in the general formulae 8, 10, 12. For this purpose, Δ of the compound of the formula 5⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾A double bond. To protect the 3-keto group, a cyclic ether is first formed for this purpose in the 3-position. Then, a⁴Isomerization of double bonds to. DELTA.⁵⁽¹⁰⁾Double bond, thereby producing the above 7 α -substituted 11 β -halo steroid, and the protecting group is cleaved again.

All of the above compounds may be further esterified or etherified if the 3-or 17 β -position has a corresponding hydroxy group. For example, compound 9 can be converted to the corresponding 17 β-ethers or 17 β -esters. One preferred compound is 11 β -fluoro-17 β - (4-sulfamoylbenzoyloxy) -7 α -methylestr-4-en-3-one. Substantially with R^17’The same groups are also suitable as C¹⁷A substituent on an oxygen-oxygen atom.

In particular, 7 α, 17 α -substituted 11 β -halo steroids of the general formulae 8, 10, 12 are suitable for the preparation of medicaments. The invention therefore relates to the use of the compounds of the general formulae 8, 10, 12 mentioned above for producing medicaments and to pharmaceutical preparations which contain at least one compound of the general formulae 8, 10, 12 mentioned above and at least one pharmaceutically compatible carrier.

The 7 α, 17 α -substituted 11 β -halo steroids of the general formulae 10, 12 according to the invention are compounds which have a strong androgenic action without the above-mentioned side effects, such as stimulation of the prostate gland, in particular non-benign prostatic hyperplasia. These compounds are easy to synthesize. It was found that the compounds of the general formula 10 or 12 of the present invention are not only useful for male HRT, but also useful as highly effective male contraceptives even without additional administration of other active ingredients if sufficient measurements are made to sufficiently lower the blood levels of LH, testosterone produced in vivo and FSH (follicle stimulating hormone). This relies on the 11 β -halo steroids of the present invention which inhibit LH and FSH release. LH stimulates the mesenchymal cells and thereby testosterone is secreted. If the blood concentration of LH is maintained at low levels, the release of endogenous testosterone also decreases. Spermatogenesis requires testosterone, while FSH stimulates sperm cells. Efficient spermatogenesis therefore requires that FSH and LH blood concentrations must be sufficiently high, whereas sufficiently high LH blood concentrations result in the release of testosterone necessary for spermatogenesis.

Since treatment with only 7 α, 17 α -substituted 11 β -halo steroids without additional sterilization active ingredients already provides an efficient male contraceptive, the administration of drugs suitable for this purpose can be greatly simplified and the cost of application can thus be significantly reduced.

The 7 α, 17 α -substituted 11 β -halo steroids of this invention may also be used in combination with a progestin to control male fertility.

Also, the 7 α, 17 α -substituted 11 β -halogen steroids of the present invention are effective in inhibiting 5 α -reductase and steroid-11-hydroxylase [ CYP11B (P450c11), g.zhang, w.l.miller, Journal of Clinical endoscopy and Metabolism, vol.81, pages3254-3256(1996) ], for example, so that irritation of the prostate can be selectively avoided and these compounds have improved pharmacokinetic properties. Inhibition of 11-hydroxylase reduces inactivation of androgen compounds and results in reduced secretion from the human body. Thus, the effectiveness of these compounds and the duration of action are improved relative to known compounds, in particular after oral administration.

For the reasons mentioned above, these compounds reduce the tropism for the 5 α -reductase and at the same time obtain the capacity for aromatization to form estrogenic steroids and have a beneficial effect on serum lipids and the central nervous system, and are particularly suitable for birth control and androgen replacement therapy.

The compounds of the general formulae 10 and 12 according to the invention were subjected to a seminal vesicle test to determine their androgenic effects and were observed to be free from the above-mentioned side effects. Uterine growth assays were performed to examine the estrogenic effects of the compounds of formula 8 of the present invention.

The 7 α, 17 α -substituted 11 β -halo steroids of the general formula 10 or 12 according to the invention or the pharmaceutical preparations according to the invention containing these compounds are outstandingly suitable for the treatment of non-sterile (non-stereo) male patients and, in principle, also of male mammals. The use of male contraception only temporarily infers male patients. After the end of the application of the active ingredient or pharmaceutical preparation according to the invention, the state is restored, so that the male patient regains reproductive capacity and spermatogenesis returns to its original level. In order to stably maintain a temporarily sterile condition for a desired period of time, the active ingredient or formulation should be continuously administered, wherein the administration is periodically repeated daily or at shorter or longer intervals depending on the administration form. After the end of one or more administrations of the active ingredient or formulation, the non-sterile condition of the male patient optionally does not recover immediately but only slowly, wherein the time interval necessary for this process depends on various factors, such as the dose, the physical state of the patient and the concurrent administration of other drugs.

If the purpose of administration includes contraception, the dose of the 7 α, 17 α -substituted 11 β -halogen steroid must be set high so that the blood concentration of LH and FSH in each case is at most 2.5I.E./ml (I.E.: International units), in particular at most 1.0I.E./ml, and the blood concentration of testosterone is at most 10nmol/l, in particular at most 3 nmol/l.

If the 7 α, 17 α -substituted 11 β -halo steroids of the present invention are used in HRT without reaching contraceptive efficacy, the dosage is set low. In this regard, it was attempted to achieve an effect level that the blood concentration of LH and FSH were each greater than 2.5i.e./ml and the blood concentration of testosterone was greater than 10 nmol/l.

The dosage of the 7 α, 17 α -substituted 11 β -halogen steroid of the general formula 10 or 12 of the present invention required for regulating the blood concentration of LH, FSH and testosterone depends on many factors and must be determined in a mode specific to administration. First, the dosage naturally depends on the kind of treatment. If the compound is to be used for male contraception, a significantly higher dose than that used in the case of HRT must be provided. The dosage will also depend on the type of 7 α, 17 α -substituted 11 β -halo steroid and its bioavailability. The mode of administration is also important with respect to the amount administered. Finally, the dosage will also depend on the physical condition of the patient to be treated or other factors, such as whether other medications are taken concurrently.

These compounds can be administered orally and parenterally, for example ip (intraperitoneal), iv (intravenous), im (intramuscular) or intradermally. They may also be implanted in tissue. The amount of these compounds administered may vary within wide limits if an effective amount is administered. The amount of compound administered may vary widely depending on the disease to be treated and the type of formulation. In humans, the daily dose is 0.1 to 100 mg. The preferred daily dosage in humans is 0.1 to 10 mg. The dosing interval depends on the intended purpose.

Dosage forms which can be used are capsules, pills, tablets, coated tablets, creams, ointments, lotions, liquid preparations such as syrups, gels, injectable liquids such as ip, iv, im or intradermal injections, etc. Thus, depending on the type, various dosage forms release the entire dose of the compound of the invention, either stepwise or over a short period of time.

For oral administration, capsules, pills, tablets, coated tablets and liquid preparations or other known oral dosage forms are used as pharmaceutical preparations. In this case, the drug may be formulated in such a way that it either releases the active ingredient over a short period of time and delivers it into the body, or has a depot effect, so that the active ingredient is fed slowly to the body over a long period of time. In addition to the 7 α, 17 α -substituted 11 β -halogen steroid, the dosage unit may contain one or more pharmaceutically compatible carriers such as agents which modify the rheological properties of the drug, surfactants, solubilizers, microcapsules, microparticles, granules, diluents, binders such as starch, sugar, sorbitol and gelatin, and fillers such as silicic acid and talc, lubricants, dyes, fragrances and others.

In particular, the 7 α, 17 α -substituted 11 β -halogen steroids of the present invention may also be formulated in the form of a solution for oral administration containing the following components in addition to the active 11 β -halogen steroid: a pharmaceutically compatible oil and/or a pharmaceutically compatible lipophilic surfactant and/or a pharmaceutically compatible hydrophilic surfactant and/or a pharmaceutically compatible water miscible solvent. In this connection, reference is made to WO-A-97/21440.

To achieve better bioavailability of the steroid, these compounds can also be formulated as cyclodextrin inclusion compounds. For this purpose, these compounds are reacted with α -, β -or γ -cyclodextrins or derivatives thereof.

If creams, ointments, lotions and liquids are used which can be applied topically, their composition must be such that the compounds of the invention can be fed to the body in sufficient quantities. In these dosage forms, there are included, for example, substances for regulating the rheological properties of drugs, surfactants, preservatives, solubilizers, diluents for increasing the permeation of the compounds of the present invention through the skin, dyes, fragrances, and adjuvants for skin care agents such as conditioning solutions (conditioners) and moisturizers. Other active ingredients may also be included in the medicament with the steroid of the invention.

For parenteral administration, the active ingredient may be dissolved or suspended in a physiologically compatible diluent. As diluents, oils are generally used, with or without solubilizers, surfactants, suspending agents or emulsifiers. Examples of oils used are olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil. For the formulation of injectable formulations, any liquid carrier in which the compounds of the present invention are soluble or emulsifiable may be used. These liquids usually contain viscosity-regulating substances, surfactants, preservatives, solubilizers, diluents and other additives by means of which the solution is made isotonic. Other active ingredients may also be administered with the 7 α, 17 α -substituted 11 β -halo steroids.

The 11 β -halogen steroids of the present invention may be administered, for example, in the form of a transdermal depot injection or implant formulation, which may be formulated in such a way that the active ingredient is slowly released. In this regard, known techniques may be used, such as reservoirs capable of dissolving or acting with the membrane. The implant may contain an inert material, such as a biodegradable polymer or a synthetic silicone such as silicone rubber. The 11 β -halogen steroids of the present invention may also be included in, for example, a patch for transdermal administration.

The following examples serve to explain the invention in more detail.

Examples

A. Microbial synthesis

11 α -hydroxy-7 α -methyl-estra-4-ene-3, 17-dione (Compound 4, B)

Example 1

A slant culture of Gnomonia cingulata strain (CBS15226) was inoculated with a medium containing 3 wt.% glucose, 1 wt.% corn steep liquor, 0.2 wt.% NaNO₃0.1% by weight of KH₂PO₄0.2% by weight of K₂HPO₄0.05 wt% KCl, 0.05 wt% MgSO₄·7H₂O and 0.002 wt% FeSO₄·7H₂1000ml of O (pH 6.0) in a 2 l Erlenmeyer flask sterilized in an autoclave for 30 minutes at 121 ℃ were shaken for 72 hours at 28 ℃ in a rotary shaker at 165 rpm. After such preculture, a 20 l fermenter covered with 19 l of sterile medium having the same final composition as the preculture described above was inoculated. In addition, 1.0ml of silicone oil and 1.0ml of synperonic (ethoxylated oxo alcohol) were additionally added to reduce foaming. After a growth phase of 12 hours at 28 ℃ under an overpressure of 0.7bar, aeration is carried out at 20 l/min with stirring at 250rpm and a solution of 4.0g of 17 β -hydroxy-7 α -methylestr-4-en-3-one in 40ml of DMF is added. Stirring and aeration were continued. After 135 hours, the culture broth was harvested, extracted with 10 liters of methyl isopropyl ketone for 12 hours, and then with 5 liters of methyl isopropyl ketone for 5 hours. The organic phases were combined and evaporated to dryness. The silicone oil was washed with hexane. Purification by silica gel chromatography in a gradient of hexane and ethyl acetate isolated 1.64g (39%) of 11 α -hydroxy-7 α -methylestra-4-ene-3, 17-dione.

Example 2

A slant culture of Glomeella cingulata strain (IFO 6425) was inoculated with a culture medium containing 3 wt% glucose, 1 wt% corn steep liquor, 0.2 wt% NaNO₃0.1% by weight of KH₂PO₄0.2% by weight of K₂HPO₄0.05 wt% KCl, 0.05 wt% MgSO₄·7H₂O and 0.002 wt% FeSO₄·7H₂1000ml of O (pH 6.0) in a 2 l Erlenmeyer flask sterilized in an autoclave for 30 minutes at 121 ℃ were shaken for 72 hours at 28 ℃ in a rotary shaker at 165 rpm. After such preculture, a 20 l fermenter covered with 19 l of sterile medium having the same final composition as the preculture described above was inoculated. In addition, 1.0ml of silicone oil and 1.0ml of synperonic were additionally added to reduce foaming. After a growth phase of 12 hours at 28 ℃ under an overpressure of 0.7bar, 2.0g of 17 β -hydroxy-7 α -methylestr-4-en-3-one in 30ml of DMF are introduced with aeration at 10 l/min, stirred at 350rpm and added. Stirring and aeration were continued. After 19 hours, the culture broth was harvested, extracted with 20 liters of methyl isopropyl ketone for 16 hours, and then with 20 liters of methyl isopropyl ketone for 23 hours. The organic phases were combined and evaporated to dryness. The residue was mostly dissolved in methanol. The silicone oil was filtered off. Concentration by evaporation and purification by silica gel chromatography in a gradient of dichloromethane and acetone isolated 1.55g (73%) of 11 α, 17 β -dihydroxy-7 α -methylestra-4-en-3-one. Recrystallizing in acetone/diisopropyl ether, and separating to obtain the product with melting point of 163 deg.C and alpha]_D＝-16°(CHCl₃C ═ 0.501) 827mg (39%) of white crystals.

Four cryo-layers (cryospheres) from a culture of Bacillus sphaericus strain (ATCC 7055) were inoculated into 500ml nutrient solutions sterilized at 121 ℃ for 30 minutes in an autoclave, containing 0.5% by weight of glucose, 0.5% by weight of yeast extract for bacteria, 0.1% by weight of peptone and 0.2% by weight of corn steep liquor (pH 7.5), and shaken at 28 ℃ for 24 hours in a rotary shaker at 165 rpm. After such preculture, each of 42 l fermenters containing 500ml of sterile medium identical to the final composition of the preculture as described above was inoculated with 10% of this culture broth. After a growth period of 4 hours at 28 ℃ in a rotary shaker at 165rpm, a solution of 50mg of 11 α, 17 β -dihydroxy-7 α -methylestra-4-en-3-one in 2.5ml of DMF was added to each flask. Stirring was continued for 48 hours. Combined cultureThe nutrient broth is extracted 2 times with 2 l of methyl isobutyl ketone. The combined organic phases were dried over sodium sulfate and evaporated to dryness. In this connection, 630mg of an oily crystalline residue were obtained. After recrystallization from acetone/diisopropyl ether, 103mg (49.2%) of a crystalline substance having a melting point of 189 ℃ and a [ alpha ] content were isolated]_D＝+40.4°(CHCl₃And c ═ 0.529) in yellow (direct crystallization without purification by chromatography as described above).

Example 3

A culture of Aspergillus ochraceus (CBS 13252) was inoculated into a medium containing 3 wt.% glucose, 1 wt.% corn steep liquor, and 0.2 wt.% NaNO₃0.1% by weight of KH₂PO₄0.2% by weight of K₂HPO₄0.05 wt% KCl, 0.05 wt% MgSO₄·7H₂O and 0.002 wt% FeSO₄·7H₂O (pH 6.0) 500ml of a nutrient solution sterilized in an autoclave at 121 ℃ for 30 minutes was placed in a 2 l Erlenmeyer flask and shaken at 28 ℃ for 72 hours in a rotary shaker at 165 rpm. After such preculture, a 10 l fermenter covered with 9.5 l of sterile medium having the same final composition as the preculture described above was inoculated. In addition, 0.5ml of silicone oil and 0.5ml of synperonic were additionally added to reduce foaming. After a growth phase of 6 hours at 28 ℃ under an overpressure of 0.7bar, 5 l/min of aeration were carried out, stirring at 350rpm and addition of 1.0g of 7 α -methylestr-4-ene-3, 17-dione in 15ml of DMF. Stirring and aeration were continued. After 22 hours, the culture broth was harvested and extracted 2 times for 4 hours with 7 liters of methyl isopropyl ketone. The organic phases were combined and evaporated to dryness. The residue was mostly dissolved in methanol. The silicone oil was filtered off. Concentration by evaporation and purification by gradient silica gel chromatography with dichloromethane and acetone gave 0.78g (74%) of 11 α -hydroxy-7 α -methylestra-4-ene-3, 17-dione. Recrystallizing in acetone/diisopropyl ether, separating to obtain alpha-alpha]_D＝+52°(CHCl₃And c-0.5905) 311mg (29.6%)。

B. Chemical preparation method

Example 4: preparation of 11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestra-4-en-3-one

a)11 beta-fluoro-7 alpha-methyl-estra-4-ene-3, 17-dione

11.5ml of perfluorobutane-1-sulfonyl fluoride were added dropwise at 0 ℃ to a solution of 13.08 g of 11 α -hydroxy-7 α -methyl-estr-4-ene-3, 17-dione (prepared according to the microbial synthesis method of [ part A ] of the invention) in 250ml of toluene and 18.2ml of 1, 8-diazabicyclo [5, 4, 0] undec-7-ene. After 1 hour, it is neutralized with 2M hydrochloric acid, added to water, extracted 4 times with ethyl acetate, washed with saturated sodium chloride solution and then concentrated by evaporation in vacuo. The crude product was purified by silica gel chromatography with hexane/ethyl acetate gradient to give 8.7 g of 11 β -fluoro-7 α -methyl-estr-4-ene-3, 17-dione.

Melting point: 101.4 deg.C, [ alpha ]]_D＝+135.8°(CHCl₃)。

b)11 β -fluoro-17 β -hydroxy-7 α -methylestra-4-en-3-one:

a solution of 8.7 g of 11 β -fluoro-7 α -methyl-estr-4-ene-3, 17-dione in 148ml of tetrahydrofuran was mixed dropwise at 0 ℃ with 29.5ml of lithium tri-tert-butoxyhydride in 1M tetrahydrofuran and stirred at 0 ℃ for 5.5 hours. Then, after addition of dilute sulfuric acid at 0 ℃, the reaction solution was added to ice water, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated by evaporation in vacuo and purified by chromatography on hexane/ethyl acetate silica gel. 5.8 g of 11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestra-4-en-3-one are obtained, having a melting point of 143-]_D＝+89.9°(CHCl₃)。

Example 5: preparation of 11 beta-fluoro-17 beta- (4-sulfamoylbenzoyloxy) -7 alpha-methylestr-4-en-3-one

500mg of 11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestr-4-en-3-one in 7.5ml pyridine solution at room temperature were mixed with 750mg of 4-sulfamoylbenzoic acid, 800mg of N, N-dicyclohexylcarbodiimideThe amine and 125mg of p-toluenesulfonic acid were mixed and stirred for 8.5 hours. It is then added to sodium bicarbonate solution, extracted 4 times with dichloromethane, washed to neutrality, dried over sodium sulfate, then concentrated by evaporation in vacuo and purified by chromatography on dichloromethane/acetone silica gel. 302mg of 11 β -fluoro-17 β - (4-sulfamoylbenzoyloxy) -7 α -methylestr-4-lin-3-one are obtained, the melting point of which is 232 ℃. [ alpha ] to]_D＝+100.5°(CHCl₃)。

Example 6: preparation of 17 alpha-ethynyl-11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestra-4-en-3-one

a)11 beta-fluoro-3-methoxy-7 alpha-methylestra-3, 5-dien-17-one

A solution of 2 g of 11 β -fluoro-7 α -methylestra-4-ene-3, 17-dione in 20ml of 2, 2-dimethoxypropane is stirred with 200mg of pyridine tosylate at 80 ℃ for 6.5 hours. It is then diluted with ethyl acetate, washed with sodium bicarbonate solution and sodium chloride solution, dried over sodium sulfate and concentrated by evaporation in vacuo. 2 g of crude 11 beta-fluoro-3-methoxy-7 alpha-methylestra-3, 5-dien-17-one are obtained.

b)17 alpha-ethynyl-11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestra-4-en-3-one

A solution of 9.17 g of cerium (III) chloride in 60ml of tetrahydrofuran was mixed dropwise at 0 ℃ with 74.2ml of an acetylene magnesium bromide (0.5M of tetrahydrofuran) solution and stirred at 0 ℃ for 1 hour. Then 2 g of a solution of 11 β -fluoro-3-methoxy-7 α -methylestra-3, 5-dien-17-one in 40ml of tetrahydrofuran are added dropwise and stirred at 0 ℃ for a further 3.5 hours. The purification step is carried out by adding saturated ammonium chloride solution, adding into water, extracting with ethyl acetate three times, washing with half concentrated hydrochloric acid, sodium bicarbonate solution and sodium chloride solution, drying with sodium sulfate, vacuum evaporating and concentrating, and purifying with hexane/ethyl acetate silica gel chromatography. 1.15 g of pure 17 α -ethynyl-11 β -fluoro-17 β -hydroxy-7 α -methylestra-4-en-3-one with a melting point of 218-220 ℃ are obtained. [ alpha ] to]_D＝+19.2°(CHCl₃)。

Example 7: preparation of 17 alpha-ethynyl-11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestra-5 (10) -en-3-one

a)3, 3-ethylenedioxy-17 alpha-ethynyl-11 beta-fluoro-7 alpha-methylestra-5 (10) -en-17 beta-ol

A solution of 700mg of 17 α -ethynyl-11 β -fluoro-17 β -hydroxy-7 α -methylestra-4-en-3-one in 7ml of dichloromethane and 4.7ml of ethylene glycol was stirred with 2.3ml of orthoformate and 30mg of p-toluenesulfonic acid hydrate for 6.5 hours at room temperature. It is then added to sodium bicarbonate solution, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated by evaporation in vacuo and chromatographed on hexane/ethyl acetate silica gel. This gave 205mg of 3, 3-ethylenedioxy-17 α -ethynyl-11 β -fluoro-7 α -methylestra-5 (10) -en-17 β -ol.

b)17 alpha-ethynyl-11 beta-fluoro-17 beta-hydroxy-7 alpha-methylestra-5 (10) -en-3-one

A solution of 205mg of 3, 3-ethylenedioxy-17 α -ethynyl-11 β -fluoro-7 α -methylestra-5 (10) -en-17 β -ol in 27ml of methanol and 3.6ml of water is stirred with 361mg of oxalic acid for 24 hours at room temperature. It is then added to sodium bicarbonate solution, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated by evaporation in vacuo and chromatographed on hexane/ethyl acetate silica gel. 95mg of 17 α -ethynyl-11 β -fluoro-17 β -hydroxy-7 α -methylestra-5 (10) -en-3-one with a melting point of 112 and 114 ℃ are obtained.

Example 8: preparation of 17 alpha-ethynyl-11 beta-fluoro-7 alpha-methylestra-1, 3, 5(10) -triene-3, 17 beta-diol

a)11 beta-fluoro-3-hydroxy-7 alpha-methylestra-1, 3, 5(10) -trien-17-one

A solution of 500mg of 11 β -fluoro-7 α -methylestra-4-ene-3, 17-dione in 16.5ml of acetonitrile is stirred with 400mg of copper (II) bromide at 25 ℃ for 6.5 hours. It is then diluted with ethyl acetate, washed with sodium bicarbonate solution and sodium chloride solution, dried over sodium sulfate, concentrated by evaporation in vacuo and purified by chromatography on hexane/acetone silica gel. 280mg of pure 11 β -fluoro-3-hydroxy-7 α -methylestra-1, 3, 5(10) -trien-17-one with a melting point of 185-186 ℃ were obtained.

b)17 alpha-ethynyl-11 beta-fluoro-7 alpha-methylestra-1, 3, 5(10) -triene-3, 17 beta-diol

A suspension of 2.03 g of cerium (III) chloride in 7.5ml of tetrahydrofuran is mixed dropwise at 0 ℃ with 16.5ml of an acetylene magnesium bromide (0.5M of tetrahydrofuran) solution and stirred at 0 ℃ for 0.5 h. 280mg of 11 β -fluoro-3-hydroxy-7 α -methylestra-1, 3, 5(10) -trien-17-one in 2.8ml of tetrahydrofuran are then added dropwise and stirred at 0 ℃ for a further 3.5 hours. The purification step is carried out by adding saturated ammonium chloride solution, adding into water, extracting with ethyl acetate four times, washing to neutrality, drying with sodium sulfate, vacuum evaporating and concentrating, and purifying with hexane/ethyl acetate silica gel chromatography.

220mg of 17 α -ethynyl-11 β -fluoro-7 α -methylestra-1, 3, 5(10) -triene-3, 17 β -diol having a melting point of 115-and 117 ℃ are obtained.

Claims (5)

1. A microbial process for the preparation of 7 α -substituted 11 α -hydroxy steroids of the general formula 4, B:

wherein R is⁷Is a P-Q group, and

p is C₁-C₄Alkylene, Q is hydrogen, C₁-C₄Alkyl or C₁-C₄A fluoroalkyl group, and a P-Q group is bonded to the steroid skeleton via P,

R¹⁰can be alpha or beta and represents H, CH₃Or CF₃And an

R¹³Is a methyl group or an ethyl group,

wherein a 7 α -substituted steroid of the general formula D is hydroxylated using a microorganism:

wherein R is⁷、R¹⁰And R¹³Having the same meaning as above, said microorganism being selected from the group consisting of: beauveria bassiana, curvularia, gibberella, plexiform shells, Japanese scale shells, macrophoma, cladosporium circinelloides, Blastomyces nigrosporus, Rhizopus and Cocephalomyces.

2. The method of claim 1, wherein the microorganism is selected from the group consisting of: beauveria bassiana, Curvularia lunata, Gibberella zeae, Rhizoctonia Malassensis, Glomerella fusarioides, Gnomonia cingulata, Haplosporia heperiana, Torulopsis piriformis, Blastomyces sphaericus and Tocopora racemosa.

3. The microbial process of claim 1 or 2, wherein R⁷Represents CH₃。

4. The microbial process of claim 1 or 2, wherein R¹⁰Represents H.

5. The microbial process of claim 1 or 2, wherein R¹³Represents CH₃。