CN1671858A - Microbial process for the preparation of 7α-substituted 11α-carboxy steroids - Google Patents

Abstract

A novel process for the preparation of precursors useful in the preparation of compounds of the general formula (8, 10, 12). In this synthesis, compounds of general formula 4, B are prepared using a microbial reaction. Radical R⁷、R¹⁰、R¹¹、R¹³、R¹⁷And R¹⁷' and U-V-W-X-Y-Z have the meanings given in the claims.

Description

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

technical field

The present invention relates to a microbial process for the preparation of 7α-substituted 11α-hydroxy steroids and to 7α,17α-substituted 11β-halogen steroids which can be prepared therefrom, to a process for the preparation of the latter compounds and to their use as well as to compounds comprising these pharmaceutical preparations. Furthermore, the present invention relates to other 7α-substituted 11β-halogen steroids, namely 7α-substituted estro-1,3,5(10)-tri alkene.

Background technique

Androgens, especially testosterone, are used to treat andropause symptoms and for male genital development and male birth control. In addition, these hormones also have some anabolic active ingredients that promote, for example, muscle growth.

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

In addition to a decrease in spermatogenesis, administration of LH-RH for male birth control also resulted in the release of LH and a decrease in testosterone levels and libido, effects that were counteracted by administration of testosterone drugs (D.E. Cummings et al., "Prostate-Sparing Effects of the Potent Androgen 7α- Methyl-19-Nortestosterone: A Potential Alternative to Testosterone for Androgen Replacement and Male Contraception,” Journal of Clinical Endocrinology and Metabolism, Vol.83, No.12, pages 4212-4219(1998)).

Combination therapy with androgenic and progestogenic active ingredients can be used to control male fertility (see for example WO 01/60376 A and references cited therein).

In the case of treatment with testosterone, it has been found that side effects develop especially prostatic hypertrophy (BPH: benign prostatic hyperplasia) due to an increase in the number of stromal cells and glands. In the 5α-reductase-regulated metabolism of progesterone, dihydroprogesterone (DHT) is formed, which in turn can lead to, for example, the development of BPH (Cummings et al., supra; WO 99/13883 A1). Therefore, BPH is treated in clinical practice by inhibition of 5α-reductase (finasteride).

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

To replace testosterone in the above-mentioned field of indications, 7α-methyl-19-nortestosterone (MeNT) was proposed, which on the one hand has a higher biological effect than testosterone due to a higher binding force to the androgen receptor , on the other hand, may hinder the metabolism of 5α-reductase due to the steric hindrance of the 7α-methyl group (Cummings et al., supra, WO 99/13883 A1, WO 99/13812 A1, US-A-5,342,834).

During the metabolism of testosterone, especially in the brain, liver and adipose tissue, a small fraction of this compound also generates estradiol through aromatization of the A ring of the steroid system. With regard to the overall function of testosterone and its metabolites, estradiol is essentially responsible for sex-specific behavior and gonadotropic regulation. Therefore, its function is thought to be beneficial in the same way that testosterone does in adult males (Cummings et al., supra).

However, the pharmacokinetics of testosterone were found to be unsatisfactory. Especially when administered orally, testosterone is excreted immediately, making the effectiveness and duration of action of the medicine prepared therefrom unsatisfactory. Therefore other derivatives of testosterone were synthesized. Such derivatives are described, for example, in US-A-5,952,319, in particular the 7α,11β-dimethyl derivative of 19nortestosterone, i.e. 7α,11β-dimethyl-17β-hydroxyestr-4-ene-3 -keto, 7α, 11β-dimethyl-17β-heptanoyloxyest-4-en-3-one, 7α, 11β-dimethyl-17β-[[(2-cyclopentylethyl)-carbonyl ]-oxy]-estr-4-en-3-one, 7α,11β-dimethyl-17β-(phenylacetoxy)-estr-4-en-3-one and 7α,11β-dimethyl Base-17β-[[(trans-4-[n-butyl]cyclohexyl)-carbonyl]-oxyl]-estr-4-en-3-one.

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

A microbial process for the synthesis of steroids is described in EP 0 900 283 B1. The paper states that est-4-ene-3,17-dione and canrenone can be converted to the corresponding 11α-hydroxyl analogs using microorganisms selected from the group consisting of: Aspergillus nigricans, Rhizopus arrhizus) and Pestelotia strains. However, reference is also made in the introduction to the specification to Shibahara et al., Biochim. Biophys. Acta, 202 (1970), 172-179, which reports that microbial 11α-hydroxylation in steroids may be unpredictable.

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

The problem on which the present invention is based is solved by the microbiological preparation of 7α-substituted steroids as claimed in claims 1, 3 and 6 and the 7α, 17α-substituted steroids as claimed in claim 11 11β-halogen steroids, methods for preparing 7α, 17α-substituted 11β-halogen steroids as claimed in claims 23, 24 and 25, 7α as claimed in claim 26, 17α-substituted Use of 11β-halogen steroids, pharmaceutical preparations containing these 7α, 17α-substituted 11β-halogen steroids as claimed in claim 27 and 7α-substituted 11β-halogenes as claimed in claim 21 Estra-1,3,5(10)-triene. Preferred embodiments of the claimed subject matter are described in the dependent claims.

definition

The following definitions refer to various parts of this specification and claims and accompanying drawing I:

All radicals, radicals or other structural units can in each case vary independently of one another within the meanings indicated.

All alkyl, alkylene, alkenyl, alkenylene, alkynyl and alkynylene groups may be straight or branched. For example, a propenyl group can be represented by one of the following chemical structures: -CH=C- _CH3 , _-CH2 -C= _CH2 , or -C( _CH3 )= _CH2 . Thus, for example methyl, ethyl, n-propyl, isopropyl n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 1-methyl Base n-pentyl, 2-methyl n-pentyl, 3-methyl n-pentyl, 4-methyl n-pentyl, 1-ethyl n-butyl, 2-ethyl n-butyl etc. all belong to C ₁ - C ₁₈ alkyl.

A cycloaliphatic alkyl group is a cycloalkyl group or a cycloalkyl group substituted by one or several alkyl groups, which are bonded directly through the cycloalkyl ring or through one of the alkyl groups.

Likewise, cycloaliphatic alkenyl is cycloalkenyl or cycloalkenyl or cycloalkyl substituted by one or more alkenyl groups or one or more alkyl groups directly through the cycloalkenyl ring or Bonding is through one of an alkenyl group or optionally an alkyl group, wherein the cycloaliphatic alkenyl group contains at least one double bond.

Aryl, on the other hand, can be phenyl, as well as 1-naphthyl or 2-naphthyl. In principle, aryl also includes heteroaryl, especially 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 formed with inorganic or organic acids, such as hydrochloric, hydrobromic, hydroiodic, acetic, citric, oxalic, tartaric and methanesulfonic acids. Especially esters with succinic acid.

The superscript number marking R, such as R ¹³ refers to its position on the steroidal ring skeleton, and the carbon atoms on the steroidal ring skeleton are numbered according to the IUPAC nomenclature. A superscript such as C ¹⁰ on the label C refers to the position of each carbon atom in the steroidal ring backbone.

Contents of the invention

Preparation of 7α-substituted 11α-hydroxy steroids of general formula 4,B using novel microbial methods:

in

R ⁷ is a PQ group, while

P is C ₁ -C ₄ alkylene, Q is hydrogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ fluoroalkyl (partially or fully fluorinated alkyl), and the PQ group passes through the P bond on the steroid skeleton,

R ¹⁰ represents H, CH ₃ or CF ₃ , and

R ¹³ is methyl or ethyl.

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

Wherein, R ⁷ , R ¹⁰ and R ¹³ have the same meaning as described in general formula 4, B, and the hydroxylation and oxidation use microorganisms selected from the following groups: Aspergillus sp., Beauveria bassiana (Beauveria sp.), Glomerella sp., Gnomonia sp., Haplosporella sp., and Rhizopus sp. Specifically preferred are Aspergillus awamori, Aspergillus fischeri, Aspergillus malignus, Aspergillus niger, Beauveria bassiana, Glomerella cingulata), Gnomoniacingulata, Haplosporella hesperedica, and Rhizopus stolonifer, among which Aspergillus awamori (CBS), Aspergillus fischeri (ATCC 1020), Aspergillus virulence (IMI 16061), Aspergillus niger (ATCC 9142) are particularly preferably used , Beauveria bassiana (ATCC 7159), Apple sorrel rot (CBS 15226, CBS 23849, CBS 98069, ATCC 56596, ATCC 64682, IFO 6425), Gnomonia cingulata (CBS 15226), Haplosporella hesperedica (CBS 20837) and grape Rhizopus cladoides (ATCC 15441).

Alternatively, the microbiological preparation method can also be carried out in two steps, wherein the hydroxylation reaction and the oxidation reaction take place in successive reaction steps. The course of the reaction can be controlled by the reaction time: by interrupting the reaction, for example after a reaction time, hexyhydroxylated but not yet oxidized species can be obtained. Therefore, the two process steps can be carried out independently or mixed fermentation.

For this purpose, the compound of general 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 sp., Aspergillus sp., Beauveria sp., Gibberella sp., Glomerella sp., Gnomonia sp., Metarrhizium sp., Nigrospora sp. ), Rhizopus sp. and Verticillium sp., which produce 7α-substituted steroids with a hydroxyl group at the 11α-position. This compound has the general formula C:

Wherein, R ⁷ , R ¹⁰ and R ¹³ have the same meanings as described in formula 4, B. Specifically used are Aspergillus malignus, Aspergillus melleus, Aspergillus niger, Aspergillus ochraceus, Beauveria bassiana, Gibberella fujikuroi ), Gibberella zeae, Glomerella cingulata, Glomerellafusaroides, Gnomonia cingulata, Metarrhizium anisopliae, Nigrospora sphaerica, Rhizopus oryzae, Rhizopus stolonifer and Verticillium dahliae. Particularly preferred in this connection for the hydroxylation are Aspergillus virulens (IMI 16061), Aspergillus melii (CBS), Aspergillus niger (ATCC 11394), Aspergillus ochraceae (NRRL 405, CBS 13252, ATCC 46504), Beauveria bassiana (ATCC 7159, IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS12736), Gibberella oryzae (ATCC 14842), Gibberella maize (CBS 4474), Phytophthora solani (ATCC 10534, CBS 23849, CBS 23749, ATCC 16646, ATCC 16052, IFO6459, IFO 6470, CBS 98069, IFO 7478, IFO 5257, ATCC 64682, ATCC15470), Glomerella fusaroides (ATCC 9552), Gnomonia cingulata (CBS15226), 5 Metarhizium anisopliae 9 Nigrospora globosa (ATCC 12772), Rhizopus oryzae (ATCC 4858, ATCC 34102, ATCC 34102), Rhizopus gluconeoides (ATCC 6227b, ATCC 15441) and Verticillium dahliae (ATCC 11405).

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 sp. (Bacillus sp.), Mycobacterium sp., Nocardia sp. and Pseudomonas sp. Concretely used are Bacillus lactimorbus, Bacillus sphaericus, Mycobacterium neoaurum, Mycobacterium smegmatis, Nocardia corallina, Nocardia microglobules ( Nocardia globerula), Nocardia minima, Nocardia restrictus, Nocardia rubropertincta, Nocardia salmonicolor, and Pseudomonas testosterone bacteria (Pseudomonas testosteroni). Among them, Bacillus lactis (ATCC 245), Bacillus sphaericus (ATCC 7055), Mycobacterium neoaurum (ATCC 9626, NRRL B-3683, NRRL B-3805), Mycobacterium smegmatis (ATCC 14468), Coral Nocardia spp. (ATCC 31338), Nocardia spp. (ATCC 9356), Nocardia spp. minimal (ATCC 19150), Nocardia spp. (NCIB 10027), Nocardia spp. rubrum (ATCC 14352 ), Nocardia salmonella (ATCC 19149), and Pseudomonas testosterone (ATCC 11996).

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

Wherein, R ⁷ , R ¹⁰ and R ¹³ have the same meanings as described in the compound of general formula 4, B. The reaction is carried out using a microorganism selected from the group consisting of Aspergillus sp., Beauveria sp., Curvularia sp., Gibberella sp., Glomerella sp., Gnomonia sp., Haplosporella sp., Helicostylum sp., Nigrospora sp., Rhizopus sp. and Syncephalastrum sp. .). Wherein the 11α-position of the steroid skeleton is hydroxylated to generate a 7α-substituted 11α-hydroxy steroid compound of the general formula 4,B. Aspergillus alliaceus, Aspergillus awamori, Aspergillus fischeri, Aspergillus malignus, Aspergillus melleus, Aspergillus nidualans, Aspergillus niger ( Aspergillus niger), Aspergillus ochraceus, Aspergillus varicolor, Beauveria bassiana, Curvularia lunata, Gibberella zeae, Apple blight Glomerella cingulata, Glomerella fusaroides, Gnomonia cingulata, Haplosporella hesperedica, Helicostylum piriformae, Nigrospora sphaerica, Rhizopus oryzae and Syncephalastrum racemosum ), of which Aspergillus cepacia (ATCC 10060), Aspergillus awamori (CBS), Aspergillus fischeri (ATCC 1020), Aspergillus virulence (IMI 16061), Aspergillus honey (CBS), Aspergillus nidulans (ATCC 11267), Aspergillus niger is particularly preferably used (ATCC 9142, ATCC11394), Aspergillus ochraceae (NRRL 405, ATCC 13252, ATCC 46504), Aspergillus versicolor (ATCC 10067), Beauveria bassiana (IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS 12736, ATCC 7159), Curvularia crescens (IX3), Gibberella zeae (CBS 4474), Apple blight (ATCC 10534, CBS 23849, CBS 23749, ATCC 16646, IFO 6459, IFO 6425, IFO 6470, ATCC 15093, ATCC 10529, IFO 5257, ATCC 56596, ATCC 64682), Glomerella fusaroides (ATCC 9552), Gnomonia cingulata (CBS 15226), Haplosporella hesperedica (CBS 20837), Cladoides piriformis (ATCC 8992), Black sporosa globosa (ATCC 12772 ), Rhizopus oryzae (ATCC 4858) and Syntocephalus racemosa (IFO 4827).

Particularly suitable is a process for the formation of 7α-substituted 11α-hydroxy steroids of the general formula 4,B, wherein independently of one another R ⁷ represents CH ₃ and/or R ¹⁰ represents H and/or R ¹³ represents CH ₃ .

The methods are carried out in a conventional manner. For this purpose, usually a sterilized nutrient solution is first prepared for the strain, and then the nutrient solution is inoculated with the strain culture solution to cultivate the strain. The preculture thus prepared is then added to a fermentor which may optionally be covered with a suitable nutrient solution. The starting material, in this case the compound of formula 3, A or the compound of formula D, is added to the fermenter preferably after the growth phase of the strain culture, so that the reaction according to the invention can be carried out. After the reaction is completed, the mixed substance is purified and isolated by conventional methods to obtain the desired 7α-substituted 11α-hydroxy steroid compound.

From the thus obtained compound of general formula 4, B, other compounds of the present invention can be synthesized by the preparation method of the present invention. Specifically, 7α, 17α-substituted 11β-halogenated steroids of general formula 8, 10, 12 and their pharmacologically compatible addition salts, esters, and amides form excellent active ingredients:

in

UVWXYZ represents one of the following ring structures: C ¹ -C ² -C ³ -C ⁴ =C ⁵ -C ¹⁰ , C ¹ -C ² -C ³ -C ⁴ -C ⁵ =C ¹⁰ , C ¹ -C ² - C ³ -C ⁴ -C ⁵ -C ¹⁰ , while in this case the oxo group (=O) is bonded to W (=C ³ ), or represents the ring structure C ¹ =C ² -C ³ = C ⁴ -C ⁵ =C ⁶ , and in this case OR ³ is bonded to W (=C ³ ),

R ³ represents H, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkanoyl or C ₃ -C ₇ cyclic ether with O atom of OR ³ group,

R ⁷ is a PQ group, and P is a C ₁ -C ₄ alkylene group, Q is hydrogen, a C ₁ -C ₄ alkyl group or a C ₁ -C ₄ fluoroalkyl group (a partially or fully fluorinated alkyl group) , and the PQ group is bonded to the steroid skeleton through P,

R ¹⁰ can be in the α- or β-position, it represents H, CH ₃ or CF ₃ , and only exists when XYZ is not C ⁴ -C ⁵ =C ¹⁰ ,

R ¹¹ is halogen,

R ¹³ is methyl or ethyl,

R ¹⁷ represents H, C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ alicyclic alkyl, C ₁ -C ₁₈ alkenyl, C ₁ -C ₁₈ alicyclic alkenyl, C ₁ -C ₁₈ alkynyl , C ₁ -C ₁₈ alkaryl, C ₁ -C ₈ alkylene nitrile or represent a PQ group, wherein the PQ group has the above-mentioned meanings,

R ¹⁷ represents H, C ₁ -C ₁₈ alkyl, C ₁ -C ₁₈ alicyclic alkyl, C ₁ -C ₁₈ alkenyl, C ₁ -C ₁₈ alicyclic alkenyl, C ₁ -C ₁₈ alkynyl , C ₁ -C ₁₈ alkaryl, wherein R ¹⁷ ' can also be bonded to 17β-oxyl through a keto group, and R ¹⁷ ' can additionally be replaced by one or more NR ¹⁸ R ¹⁹ groups or one or more Substituted by SO _x R ²⁰ , where x=0, 1 or 2, and R ¹⁸ , R ¹⁹ and R ²⁰ have in each case independently of one another the meaning of R ¹⁷ .

These compounds, which are obtainable by an additional process step from 7α-substituted 11α-hydroxy steroids of the general formula 4,B, are valuable active ingredients with a strong androgenic effect without the aforementioned side effects. These compounds are suitable for the preparation of medicaments, in particular effective contraceptives and active ingredients for hormone replacement therapy (HRT).

If R ^17' is additionally substituted by NR ¹⁸ R ¹⁹ , it can be methylamino, dimethylamino, ethylamino, diethylamino, cyclohexylamino, dicyclohexylamino, phenylamino, diphenylamino , benzylamino or dibenzylamino.

Particularly suitable 7α,17α-substituted 11β-halogen steroids having the general formulas 8, 10, 12 are those wherein UVWXYZ represents the ring structure C ¹ -C ² -C ³ -C ⁴ =C ⁵ -C ¹⁰ , C A compound of ¹ -C ² -C ³ -C ⁴ -C ⁵ =C ¹⁰ or C ¹ =C ² -C ³ =C ⁴ -C ⁵ =C ¹⁰ .

The first case (UVWXYZ stands for C ¹ -C ² -C ³ -C ⁴ =C ⁵ -C ¹⁰ ) is a steroid compound of general formula 10:

The second case (UVWXYZ stands for C ¹ -C ² -C ³ -C ⁴ -C ⁵ =C ¹⁰ ) is a steroid compound of general formula 12:

Compounds of general formulas 10 and 12 are androgenic compounds.

The third case (UVWXYZ stands for C ¹ =C ² -C ³ =C ⁴ -C ⁵ =C ⁶ ) is a steroid compound of general formula 8:

These compounds are estrogens (estrogen receptor binding compounds).

In the above three cases, R ³ , R ⁷ , R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁷ and R ^17′ have the same meanings as the corresponding groups in formulas 8, 10 and 12.

Independently of each other, preferably, R ¹ represents H and/or R ⁷ represents CH ₃ and/or R ¹¹ represents fluorine and/or R ¹³ represents CH ₃ and/or R ¹⁷ represents H, CH ₃ , especially ethynyl C ₁ -C ₁₈ alkynyl, CH ₂ CN or CF ₃ and/or R ^17′ represents H.

Among the 7α, 17α-substituted 11β-halogen steroids of the general formulas 8, 10, 12 which are particularly suitable for the present invention are:

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

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

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

These compounds can be prepared using the following methods:

For the preparation of 7α,17α-substituted 11β-halogen steroids of general formula 10 in which UVWXYZ represents the ring structure C ¹ -C ² -C ³ -C ⁴ =C ⁵ -C ¹⁰ , the microbiological preparation by the present invention is used The 7α-substituted 11α-hydroxy steroids of the general formula 4,B obtained by the process are used as starting materials.

In the first step of the synthesis, these thus obtained 7α-substituted 11α-hydroxy steroids were converted to the corresponding 7α-substituted 11β-halogen steroids by nucleophilic substitution using a halogenated dehydroxylation reagent 5:

All compounds commonly used for this purpose can be used as halodehydroxylation reagents, such as hydrofluoric, hydrochloric, hydrobromic or hydroiodic acids, thionyl chloride or bromide, phosphorus pentachloride, phosphorus Acyl chloride, N-chlorosuccinimide, triphenylphosphine/carbon tetrachloride, HF/pyridine or diethylaminosulfur trifluoride or preferably nonaflyl fluoride/1, 5-diazabicyclo[5.4.0]undecene.

Compound 10 was then prepared from compound 5 by selective alkylation of ring backbone ^C17 (see Figure 1). Common alkylating agents can be used for the selective alkylation, such as Grignard compounds and organometallic compounds, especially alkyllithium compounds. Ethynylmagnesium bromide, for example, can be used as an alkylating agent to prepare the corresponding 17α-ethynyl-17β-hydroxyestr-4-en-3-one from est-4-ene-3,17-dione.

Compounds of general formula 10 can be used and isomerized to isomerize the Δ ⁴ double bond into a Δ ⁵⁽¹⁰⁾ double bond, which can be used to prepare wherein UVWXYZ represents the ring structure C ¹ -C ² -C ³ -C ⁴ - ^C5 = ^C10 7α,17α-substituted 11β-halogen steroids of general formula 12. To protect the 3-keto group, a cyclic ether is first generated for this purpose in the 3-position. Then, the ^Δ4 double bond is isomerized to the Δ5 ⁽¹⁰⁾ double bond, resulting in 7α,17α-substituted 11β-halogen steroids of general formula 12, and the protecting group is cleaved again.

To prepare additional 7α,17α-substituted 11β-halogen steroids of structure 8, wherein UVWXYZ represents the ring structure C ¹ =C ² -C ³ =C ⁴ -C ⁵ =C ¹⁰ , the procedure is as follows:

First, the 7α-substituted 11α-hydroxy steroids of the general formula 5 prepared by microbiological hydroxylation and oxidation of the general formula 4, B were prepared by halogen dehydroxylation in a nucleophilic substitution reaction as described above. The corresponding 11β-halogen steroids.

Oxidation of the former by e.g. copper(II) salts yields 7α-substituted estra-1,3,5(10)-trienes of general formula 6:

The meanings of R ³ , R ⁷ , R ¹¹ and R ¹³ are as above. If R ³ represents H, these compounds can be directly synthesized. If another group replaces H in R ³ , the corresponding ether or ester must be prepared by known methods after oxidation to the 1,3,5(10)-triene ring.

Since the 7α-substituted 11β-haloestra-1,3,5(10)-triene having the general formula 6 and its pharmaceutically compatible addition salts, esters and amides are novel, therefore, in this Protection is claimed in the invention as an intermediate in the synthesis of 7α,17α-substituted 11β-halogen steroids of general formula 8.

A particularly preferred 7α-substituted 11β-haloestra-1,3,5(10)-triene of general formula 6 is 11β-fluoro-3-hydroxy-7α-methylestra-1,3,5 (10)-Trien-17-one.

The 7α, 17α-substituted 11β-halogen steroids of the present invention can be prepared from the 7α-substituted 11β-halogenestra-1,3,5(10)-triene of the general formula 6, the method The same method as described above for the preparation of general formula 10 by selective oxidation of ring backbone ^C17 .

In addition, 7α-substituted 11β-halogen steroids of general formula 9 can also be prepared by microbiological hydroxylation and oxidation of general formula 4, B prepared by general formula 3, A or D, and said 7α-substituted 11β-halogen steroids are also androgenic:

Wherein, the meanings of R ⁷ , R ¹¹ and R ¹³ are as above. A particularly preferred compound is 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one. The compounds of general formula 9 and their pharmaceutically compatible addition salts, esters and amides also have androgenic effects.

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

Furthermore, compounds of general formula 9 can be converted to the corresponding 7α-substituted 11β-halogenestr-5(10)-enes:

Wherein, the meanings of R ⁷ , R ¹⁰ , R ¹¹ and R ¹³ are the same as those described in Formulas 8, 10 and 12. For this purpose, the ^Δ4 double bond of the compound of general formula 9 is isomerized to a ^Δ5(10) double bond. To protect the 3-keto group, a cyclic ether is first generated for this purpose in the 3-position. Then, the ^Δ4 double bond is isomerized to the ^Δ5(10) double bond, resulting in a 7α-substituted 11β-halogen steroid, and the protecting group is cleaved again.

Finally, the corresponding 7α-substituted 11β-halogenestr-5(10)-enes can be converted from compounds of general formula 5 by isomerizing the ^Δ4 double bond to the ^Δ5(10) double bond.

Wherein, the meanings of R ⁷ , R ¹⁰ , R ¹¹ and R ¹³ are the same as those described in Formulas 8, 10 and 12. For this purpose, the ^Δ4 double bond of the compound of general formula 5 is isomerized to a ^Δ5(10) double bond. To protect the 3-keto group, a cyclic ether is first generated for this purpose in the 3-position. Then, the ^Δ4 double bond is isomerized to the Δ5 ⁽¹⁰⁾ double bond, thereby generating the above-mentioned 7α-substituted 11β-halogen steroid, and the protecting group is cleaved again.

All of the above compounds can be further esterified or etherified if the 3- or 17β-position has a corresponding hydroxyl group. For example, compound 9 can be converted to the corresponding 17[beta]-ether or 17[beta]-ester. A preferred compound is 11β-fluoro-17β-(4-sulfamoylbenzoyloxy)-7α-methylestr-4-en-3-one. Essentially the same groups as described for R ^17' are also suitable as substituents on the C ¹⁷ oxygen-oxygen atom.

In particular, the 7[alpha],17[alpha]-substituted 11[beta]-halogen steroids of the general formulas 8, 10, 12 are suitable for the preparation of medicaments. The present invention therefore relates to the use of the compounds of the above general formulas 8, 10 and 12 in the preparation of medicines and pharmaceutical preparations containing at least one compound of the above general formulas 8, 10 and 12 and at least one pharmaceutically compatible carrier.

The 7α, 17α-substituted 11β-halogenosteroids of the general formulas 10, 12 of the present invention are compounds having strong androgenic effects without the aforementioned side effects such as stimulation of the prostate gland (especially non-benign prostatic hyperplasia). These compounds are easy to synthesize. It has been found that these compounds of formula 10 or 12 of the present invention are not only useful for HRT in men, but if measured sufficiently to sufficiently lower blood concentrations of LH, testosterone produced in vivo, and FSH (follicle-stimulating hormone), even without additional administration In the absence of other active ingredients these compounds are also useful as highly effective male contraceptives. This relies on the 11β-halogenosteroids of the present invention that inhibit the release of LH and FSH. LH stimulates interstitial cells to secrete testosterone. If the blood concentration of LH is maintained at low levels, the release of endogenous testosterone also decreases. Testosterone is required for spermatogenesis, and FSH stimulates sperm cells. Efficient spermatogenesis therefore requires sufficiently high blood concentrations of FSH and LH, and sufficiently high blood concentrations of LH result in the release of testosterone necessary for spermatogenesis.

Since treatment with only 7α,17α-substituted 11β-halogen steroids without the use of additional sterilizing active ingredients can provide a highly effective method of male contraception, the administration of drugs suitable for this purpose can be greatly simplified, and the cost of application can be significantly reduced by this.

The 7α,17α-substituted 11β-halogen steroids of the present invention can also be used in combination with progestogens to control male fertility.

And, 7α of the present invention, 17α-substituted 11β-halogen steroids can effectively inhibit 5α-reductase and steroid-11-hydroxylase [CYP11B (P450c11), G.Zhang, W.L.Miller, Journal of Clinical Endocrinology and Metabolism, Vol.81, pages 3254-3256 (1996)], so that, for example, stimulation of the prostate can be selectively avoided and these compounds have improved pharmacokinetic properties. Inhibition of 11-hydroxylase reduces the inactivation of androgenic compounds and leads to a decrease in their secretion from the human body. Thus, the effectiveness and duration of action of these compounds is improved compared to known compounds, especially after oral administration.

For the above reasons, these compounds reduce the tropism for 5α-reductase and at the same time acquire the ability to aromatize estrogenic steroids and have beneficial effects on serum lipids and central nervous system, especially for birth control and androgen replacement therapy .

Seminal vesicle tests were carried out on the compounds of general formulas 10 and 12 of the present invention to determine their androgenic effects and to observe that they did not appear the above-mentioned side effects. A uterine growth test was carried out to detect the estrogen effect of the compound of general formula 8 of the present invention.

The 7α, 17α-substituted 11β-halogen steroids of the general formula 10 or 12 of the present invention or the pharmaceutical preparations comprising these compounds of the present invention are extremely suitable for the treatment of non-sterile (non-sterile) male patients, basically also suitable for male mammal. The use of male contraception only renders male patients temporarily sterile. After the application of the active ingredient or pharmaceutical preparation of the present invention ends, it returns to the original state, so that the male patient recovers his reproductive ability, and the spermatogenesis returns to the original level. In order to stabilize the condition of temporary infertility for the desired period, the active ingredient or preparation should be administered continuously, wherein the administration is repeated daily or periodically at shorter or longer intervals, depending on the form of administration. After one or repeated administration of the active ingredient or preparation, 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 patient's physical state, and other drugs administered in parallel.

If the purpose of administration includes contraception, the dose of the 7α,17α-substituted 11β-halogenosteroid must be set high so that the blood concentrations of LH and FSH are in each case up to 2.5 I.E./ml (I.E.: International Units), especially up to 1.0 I.E./ml, and a blood concentration of testosterone up to 10 nmol/l, especially up to 3 nmol/l.

If the 7[alpha],17[alpha]-substituted 11[beta]-halogenosteroids of the present invention are used for HRT without achieving a contraceptive effect, the dose is set to be low. In this connection, an attempt was made to achieve an effect level which would result in a blood concentration of LH and FSH greater than 2.5 I.E./ml each and a blood concentration of testosterone greater than 10 nmol/l.

Dosages of 7α,17α-substituted 11β-halogenosteroids of formula 10 or 12 of the present invention for regulation of LH, FSH and testosterone blood concentrations depend on many factors and must therefore be determined in a specific mode of administration. Firstly, the dosage naturally depends on the kind of treatment. If the compound is used for male contraception, significantly higher doses must be provided than in the case of HRT. The dose also depends on the type of 7α,17α-substituted 11β-halogen steroid and its bioavailability. The mode of administration is also very important for the amount administered. Finally, the dosage also depends on the physical fitness of the patient to be treated or other factors, such as whether other drugs are taken concurrently.

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

Applicable dosage forms are capsules, pills, tablets, coated tablets, creams, ointments, lotions, liquid preparations such as syrups, gels, injectable liquids such as ip, iv, im or intradermal injection and the like. Thus, depending on the type, the various dosage forms release the entire dose of the compound of the invention gradually 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 can 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 it has a depot effect, allowing it to slowly deliver the active ingredient to the body over a long period of time. Element. In addition to 7α,17α-substituted 11β-halogen steroids, the dosage unit may contain one or more pharmaceutically compatible carriers, such as substances that adjust 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 other substances.

In particular, the 7α, 17α-substituted 11β-halogenosteroids of the present invention can also be formulated in the form of a solution for oral administration, which contains the following ingredients in addition to the active 11β-halogenosteroids: pharmaceutically compatible Sexual oils and/or pharmaceutically compatible lipophilic surfactants and/or pharmaceutically compatible hydrophilic surfactants and/or pharmaceutically compatible water-miscible solvents. In this regard, reference is made to WO-A-97/21440.

In order to achieve better bioavailability of steroids, these compounds can also be formulated as cyclodextrin inclusion complexes. For this purpose, these compounds are reacted with α-, β- or γ-cyclodextrins or derivatives thereof.

If topical creams, ointments, lotions and liquids are employed, their composition must be such that the compounds of this invention are administered to the body in sufficient quantities. In these dosage forms, for example, substances that adjust the rheological properties of the drug, surfactants, preservatives, solubilizers, diluents, substances that increase the penetration ability of the compound of the present invention through the skin, dyes, fragrances, and substances such as conditioning fluids ( Conditioner) and moisturizing lotion skin care agent accessories. Other active ingredients may also be included in the drug together with the steroids of the invention.

For parenteral administration, the active ingredient may be dissolved or suspended in a physiologically compatible diluent. As diluents, oils are generally employed, 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 preparation of injectables, any liquid carrier in which the compound of this invention can be dissolved or emulsified can be used. These liquids usually contain viscosity-adjusting substances, surfactants, preservatives, solubilizers, diluents and other additives by which the solution is made isotonic. Other active ingredients can also be administered together with the 7α,17α-substituted 11β-halogen steroids.

The 11β-halogenosteroids of the invention may be administered, for example, in the form of transdermal depot injections or implants, which may be formulated so as to provide slow release of the active ingredient. In this regard, known techniques can be used, such as depots capable of dissolving or cooperating with membranes. Implants may contain inert materials such as biodegradable polymers or synthetic silicones such as silicone rubber. The 11[beta]-halogenosteroids of the present invention may also be included, for example, in a patch for transdermal administration.

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

Example

A. Microbial Synthesis

11α-hydroxy-7α-methyl-estr-4-ene-3,17-dione (compound 4, B)

Example 1

The inclined bacillus culture of Gnomonia cingulata strain (CBS 15226) was inoculated in a container containing 3% by weight of glucose, 1% by weight of corn steep liquor, 0.2% by weight of NaNO ₃ , 0.1% by weight of KH ₂ PO ₄ , 0.2% by weight of K ₂ HPO ₄ , 0.05% by weight of KCl, 0.05% by weight of MgSO ₄ ·7H ₂ O and 0.002% by weight of FeSO ₄ ·7H ₂ O (pH 6.0) of 1000 ml of 2 liters of Erlenmeyer nutrient solution sterilized at 121° C. for 30 minutes in an autoclave The flask was shaken on a rotary shaker at 165 rpm for 72 hours at 28°C. After such preculture, it was inoculated in a 20 liter fermenter covered with 19 liters of sterile medium having the same final composition as the preculture described above. In addition, another 1.0 ml of silicone oil and 1.0 ml of synperonic (oxoalcoholethoxylate) were added to reduce foaming. After a 12-hour growth period at an overpressure of 0.7 bar at a temperature of 28°C, aeration at 20 l/min, stirring at 250 rpm and addition of 4.0 g of 17β-hydroxy-7α-methylestr-4-en-3-one 40ml DMF solution. Continue to stir and aerate. After 135 hours, the culture broth was harvested and 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. Wash away the silicone oil with hexane. Purification by silica gel chromatography with a gradient of hexane and ethyl acetate isolated 1.64 g (39%) of 11α-hydroxy-7α-methylestr-4-ene-3,17-dione.

Example 2

Inoculate the inclined bacillus culture of Glomerella cingulata strain (IFO 6425) in a container containing 3% by weight of glucose, 1% by weight of corn steep liquor, 0.2% by weight of NaNO ₃ , 0.1% by weight of KH ₂ PO ₄ , 0.2% by weight of K ₂ HPO ₄ , 0.05% by weight of KCl, 0.05% by weight of MgSO ₄ ·7H ₂ O and 0.002% by weight of FeSO ₄ ·7H ₂ O (pH 6.0) of 1000 ml of 2 liters of Erlenmeyer nutrient solution sterilized at 121° C. for 30 minutes in an autoclave The flask was shaken on a rotary shaker at 165 rpm for 72 hours at 28°C. After such preculture, it was inoculated in a 20 liter fermenter covered with 19 liters of sterile medium having the same final composition as the preculture described above. In addition, another 1.0ml of silicone oil and 1.0ml of synperonic were added to reduce foaming. After a 12-hour growth period at an overpressure of 0.7 bar at a temperature of 28°C, aeration at 10 l/min, stirring at 350 rpm and addition of 2.0 g of 17β-hydroxy-7α-methylestr-4-en-3-one 30ml of DMF solution. Continue to stir and aerate. After 19 hours, the culture broth was harvested and extracted with 20 liters of methyl isopropyl ketone for 1 hour 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. Filter off the silicone oil. Concentration by evaporation and chromatography on silica gel with a gradient of dichloromethane and acetone yielded 1.55 g (73%) of 11α,17β-dihydroxy-7α-methylestr-4-en-3-one isolated. After recrystallization from acetone/diisopropyl ether, 827 mg (39%) of white crystals with melting point 163°C, [α] _D = -16° (CHCl ₃ , c = 0.501) were isolated.

Four cryospheres (cryosphere) derived from the culture of Bacillus sphaericus bacterial strain (ATCC 7055) were inoculated in a container containing 0.5% by weight of glucose, 0.5% by weight of bacterial yeast extract, 0.1% by weight of peptone and 0.2% by weight of corn steep liquor. (pH 7.5) in 500 ml of nutrient solution autoclaved at 121° C. for 30 minutes in a 2 liter Erlenmeyer flask and shaken at 28° C. on a rotary shaker at 165 rpm for 24 hours. After such preculture, each of four 2-liter fermenters containing 500 ml of a sterile medium having the same final composition as the preculture described above was inoculated with 10% of the culture broth. After a 4-hour growth period at 28°C in a rotary shaker at 165 rpm, 50 mg of 11α, 17β-dihydroxy-7α-methylestr-4-en-3-one was added to each flask at 2.5 ml solution in DMF. Stirring was continued for 48 hours. The combined culture broths were extracted twice with 2 liters of methyl isobutyl ketone. The combined organic phases were dried over sodium sulfate and evaporated to dryness. In this connection, 630 mg of an oily crystalline residue are obtained. After recrystallization from _acetone /diisopropyl ether, 103 mg (49.2%) of yellow crystals (directly crystallized without _passing through aforementioned chromatographic purification).

Example 3

Inoculate the culture of Aspergillus ochrescens (CBS 13252) in a culture chamber containing 3% by weight of glucose, 1% by weight of corn steep liquor, 0.2% by weight of NaNO ₃ , 0.1% by weight of KH ₂ PO ₄ , 0.2% by weight of K ₂ HPO ₄ , 0.05% by weight KCl, 0.05% by weight MgSO ₄ 7H ₂ O and 0.002% by weight FeSO ₄ 7H ₂ O (pH 6.0) 500ml of nutrient solution sterilized at 121° C. for 30 minutes in an autoclave 2 Liter Erlenmeyer flasks were shaken at 28 °C for 72 h at 165 rpm on a rotary shaker. After such preculture, it was reinoculated in a 10-liter fermentor covered with 9.5 liters of sterile medium having the same final composition as the preculture described above. In addition, another 0.5ml of silicone oil and 0.5ml of synperonic were added to reduce foaming. After a 6-hour growth period at an overpressure of 0.7 bar and a temperature of 28°C, aeration at 5 liters/min, stirring at 350 rpm and addition of 1.0 g of 7α-methylestr-4-ene-3,17-dione 15ml DMF solution. Continue to stir and aerate. After 22 hours, the culture broth was harvested and extracted twice with 7 liters of methyl isopropyl ketone for 4 hours. The organic phases were combined and evaporated to dryness. The residue was mostly dissolved in methanol. Filter off the silicone oil. Concentration by evaporation and chromatography on silica gel with a gradient of dichloromethane and acetone yielded 0.78 g (74%) of 11α-hydroxy-7α-methylestr-4-ene-3,17-dione isolated. After recrystallization from acetone/diisopropyl ether, 311 mg (29.6%) of white crystals with melting point 200° C., [α] _D =+52° (CHCl ₃ , c=0.5905) were isolated.

B. Chemical preparation method

Example 4: Preparation of 11β-fluoro-17β-hydroxyl-7α-methylestr-4-en-3-one

a) 11β-fluoro-7α-methyl-estr-4-ene-3,17-dione

11.5 ml of perfluorobutane-1-sulfonyl fluoride was added dropwise at 0° C. to 13.08 g of 11α-hydroxy-7α-methyl-estr-4-ene-3,17-dione (according to the present invention [part A] prepared by microbial synthesis) in 250ml of toluene and 18.2ml of 1,8-diazabicyclo[5,4,0]undec-7-ene in solution. After 1 hour, it was neutralized with 2M hydrochloric acid, added to water, extracted 4 times with ethyl acetate, washed with saturated sodium chloride solution, and concentrated by evaporation to dryness in vacuo. The crude product was purified by silica gel chromatography with a hexane/ethyl acetate gradient to yield 8.7 g of 11β-fluoro-7α-methyl-estr-4-ene-3,17-dione. Melting point: 101.4°C, [α] _D =+135.8° (CHCl ₃ ).

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

A solution of 8.7 g of 11β-fluoro-7α-methyl-estr-4-ene-3,17-dione in 148 ml of tetrahydrofuran was mixed dropwise with 29.5 ml of 1M tetrahydrofuran at 0 °C with tri-tert-butoxy lithium aluminum hydride Mix and stir at 0°C for 5.5 hours. Then, after dilute sulfuric acid was added at 0°C, the reaction solution was added to ice water, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated by vacuum evaporation and purified by hexane/ethyl acetate silica gel chromatography. 5.8 g of 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one are obtained, melting point 143-144° C., [α] _D =+89.9° (CHCl ₃ ).

Example 5: Preparation of 11β-fluoro-17β-(4-sulfamoylbenzoyloxy)-7α-methylestr-4-en-3-one

Carbonate 500mg of 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one in 7.5ml of pyridine with 750mg of 4-sulfamoylbenzoic acid and 800mg of N,N-dicyclohexyl at room temperature The diimine and 125 mg 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 until neutral, dried over sodium sulfate, concentrated by evaporation in a vacuum and purified by chromatography on silica gel with dichloromethane/acetone. This gave 302 mg of 11β-fluoro-17β-(4-sulfamoylbenzoyloxy)-7α-methylestr-4-en-3-one of melting point 232°C. [α] _D = +100.5° (CHCl ₃ ).

Example 6: Preparation of 17α-ethynyl-11β-fluoro-17β-hydroxyl-7α-methylestr-4-en-3-one

a) 11β-fluoro-3-methoxy-7α-methylestr-3,5-dien-17-one

A solution of 2 g of 11β-fluoro-7α-methylestr-4-ene-3,17-dione in 20 ml of 2,2-dimethoxypropane was stirred with 200 mg of pyridine tosylate at 80°C 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 a vacuum. 2 g of crude 11β-fluoro-3-methoxy-7α-methylestr-3,5-dien-17-one were obtained.

b) 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one

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

Example 7: Preparation of 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-5(10)-en-3-one

a) 3,3-Ethylenedioxy-17α-ethynyl-11β-fluoro-7α-methylestr-5(10)-en-17β-ol

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

b) 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-5(10)-en-3-one

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

Example 8: Preparation of 17α-ethynyl-11β-fluoro-7α-methylestra-1,3,5(10)-triene-3,17β-diol

a) 11β-fluoro-3-hydroxy-7α-methylestr-1,3,5(10)-trien-17-one

A solution of 500 mg of 11β-fluoro-7α-methylestr-4-ene-3,17-dione in 16.5 ml of acetonitrile was stirred with 400 mg of copper(II) bromide at 25° C. 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 silica gel with hexane/acetone. This gives 280 mg of pure 11β-fluoro-3-hydroxy-7α-methylestra-1,3,5(10)-trien-17-one with a melting point of 185-186°C.

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

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

This gives 220 mg of 17α-ethynyl-11β-fluoro-7α-methylestra-1,3,5(10)-triene-3,17β-diol with a melting point of 115-117° C.

Claims (27)

1, preparation general formula 4, the microbial process of 7 α of B-substituted 11 Alpha-hydroxy steroides:

Wherein, R ⁷Be the P-Q base, and

P is C ₁-C ₄Alkylidene group, Q are hydrogen, C ₁-C ₄Alkyl or C ₁-C ₄Fluoro-alkyl,

And the P-Q base is bonded on the steroid backbone by P,

R ¹⁰Can be α position or β position, and represent H, CH ₃Or CF ₃, and

R ¹³Be methyl or ethyl,

Wherein use the microorganism be selected from aspergillus, muscardine, sick mould, sundial shell, big monospore and head mold with general formula 3,7 α of A-substituted steroide hydroxylation and oxidation:

Wherein, R ⁷, R ¹⁰And R ¹³Has implication same as described above.

2, the method for claim 1 is characterized in that this microorganism is selected from following group: Aspergillus awamori, expense Xi Shi aspergillus, poisonous aspergillus, black aspergillus, silkworm muscardine, apple withered rotten disease are mould, Gnomonia cingulata, Haplosporella hesperedica and Rhizopus stolonifer.

3, preparation general formula 4, the microbial process of 7 α of B-substituted 11 Alpha-hydroxy steroides:

Wherein, R ⁷Be the P-Q base, and

P is C ₁-C ₄Alkylidene group, Q are hydrogen, C ₁-C ₄Alkyl or C ₁-C ₄Fluoro-alkyl,

And the P-Q base is bonded on the steroid backbone by P,

R ¹⁰Can be α position or β position, and represent H, CH ₃Or CF ₃, and

R ¹³Be methyl or ethyl,

Wherein, general formula 3,7 α of A-substituted steroide

R wherein ⁷, R ¹⁰And R ¹³Have implication same as described above,

It uses the first microorganism hydroxylation that is selected from aspergillus, muscardine, red mould, sick mould, sundial shell, green muscardine fungus, black pore fungi, head mold and wheel branch spore at 11 alpha-positions in the first microbial process step, generate 7 α-substituted 11 Alpha-hydroxy steroides of general formula C:

R wherein ⁷, R ¹⁰And R ¹³Have implication same as described above,

Then, 7 α-substituted 11 Alpha-hydroxy steroides with the general formula C that generated in the second microbial process step use second microbiological oxidation that is selected from genus bacillus, mycobacterium, Nocardia bacteria and pseudomonas, generate general formula 4,7 α of B-substituted steroide.

4, method as claimed in claim 3, wherein said first microorganism is selected from following group: poisonous aspergillus, honey aspergillus, black aspergillus, reddish brown aspergillus, silkworm muscardine, rice dislike that red mould, the Gibberella zeae of seedling, apple withered rotten disease are mould, Glomerella fusaroides, Gnomoniacingulata, Metarhizium anisopliae, nigrospora sphaerica, Rhizopus oryzae, Rhizopus stolonifer and a Garden Dahlia wheel branch spore.

5, as claim 3 or 4 described methods, wherein said second microorganism is selected from following group: bacillus lentimorbus, Bacillus sphaericus, Mycobacterium neoaurum, M. smegmatics, coral Nocardia bacteria, globule nocardia, minimum Nocardia bacteria, qualification Nocardia bacteria, scarlet Nocardia bacteria, salmon look Nocardia bacteria and testosterone pseudomonas.

6, preparation general formula 4, the microbial process of 7 α of B-substituted 11 Alpha-hydroxy steroides:

Wherein, R ⁷Be the P-Q base, and

P is C ₁-C ₄Alkylidene group, Q are hydrogen, C ₁-C ₄Alkyl or C ₁-C ₄Fluoro-alkyl,

And the P-Q base is bonded on the steroid backbone by P,

R ¹⁰Can be α position or β position, and represent H, CH ₃Or CF ₃, and

R ¹³Be methyl or ethyl,

Wherein use the 7 αs-substituted steroide hydroxylation of microorganism with general formula D:

Wherein, R ⁷, R ¹⁰And R ¹³Have implication same as described above, described microorganism is selected from following group: aspergillus, muscardine, curved spore, red mould, sick mould, sundial shell, big monospore, mould, the black spore of convolution branch, head mold and mould altogether.

7, method as claimed in claim 6, wherein said microorganism are selected from following group: onion aspergillus, Aspergillus awamori, expense Xi Shi aspergillus, poisonous aspergillus, honey aspergillus, Aspergillus nidulans, black aspergillus, reddish brown aspergillus, aspergillus versicolor, silkworm muscardine, new long-radius elbow spore, Gibberella zeae, apple withered rotten disease are mould, Glomerella fusaroides, Gnomonia cingulata, Haplosporellahesperedica, pyriform convolution branch are mould, nigrospora sphaerica, Rhizopus oryzae and racemose gongtou mould.

8, as the described microbial process of one of claim 1 to 7, wherein R ⁷Represent CH ₃

9, as the described microbial process of one of claim 1 to 8, wherein R ¹⁰Represent H.

10, as the described microbial process of one of claim 1 to 9, wherein R ¹³Represent CH ₃

11, general formula 8,10,12 7 α, and 17 α-substituted 11 beta-halogen steroides and their pharmacology consistency additive salt, ester and acid amides:

Wherein, U-V-W-X-Y-Z represents one of following ring structure: C ¹-C ²-C ³-C ⁴=C ⁵-C ¹⁰, C ¹-C ²-C ³-C ⁴-C ⁵=C ¹⁰, C ¹-C ²-C ³-C ⁴-C ⁵-C ¹⁰, in the case, the oxo base (=O) be bonded to W (=C ³) on, perhaps represent ring structure C ¹=C ²-C ³=C ⁴-C ⁵=C ⁶, in the case, OR ³Be bonded to W (=C ³) on.

R ³Represent H, C ₁-C ₄Alkyl, C ₁-C ₄Alkyloyl or have OR ³The C of the O atom of base ₃-C ₇Cyclic ethers,

R ⁷Be the P-Q base, and P is C ₁-C ₄Alkylidene group, Q are hydrogen, C ₁-C ₄Alkyl or C ₁-C ₄Fluoro-alkyl, and the P-Q base is bonded on the steroid backbone by P,

R ¹⁰Can be in α-or β-position, it represents H, CH ₃Or CF ₃, and be not C at X-Y-Z only ⁴-C ⁵=C ¹⁰In time, exist,

R ¹¹Be halogen,

R ¹³Be methyl or ethyl,

R ¹⁷Represent H, C ₁-C ₁₈Alkyl, C ₁-C ₁₈Alicyclic alkyl, C ₁-C ₁₈Thiazolinyl, C ₁-C ₁₈Alicyclic thiazolinyl, C ₁-C ₁₈Alkynyl, C ₁-C ₁₈Alkaryl, C ₁To C ₈The alkylidene group nitrile, or represent the P-Q base, wherein the P-Q base has above-mentioned implication,

R ¹⁷' represent H, C ₁-C ₁₈Alkyl, C ₁-C ₁₈Alicyclic alkyl, C ₁-C ₁₈Thiazolinyl, C ₁-C ₁₈Alicyclic thiazolinyl, C ₁-C ₁₈Alkynyl or C ₁-C ₁₈Alkaryl, wherein R ¹⁷' also can be bonded to 17 β-oxygen base by ketone group, and R ¹⁷' also can add by one or more NR ¹⁸R ¹⁹Base or one or more SO _xR ²⁰Replace, wherein, x is 0,1 or 2, and R ¹⁸, R ¹⁹And R ²⁰Has R in all cases independently of one another ¹⁷Implication.

12,7 α as claimed in claim 11,17 α-substituted 11 beta-halogen steroides, wherein U-V-W-X-Y-Z represents ring structure C ¹-C ²-C ³-C ⁴=C ⁵-C ¹⁰Or C ¹-C ²-C ³-C ⁴-C ⁵=C ¹⁰Or C ¹=C ²-C ³=C ⁴-C ⁵=C ¹⁰

13, as claim 11 or 12 described 7 α, 17 α-substituted 11 beta-halogen steroide, wherein R ¹Represent H.

14, as described 7 α of one of claim 11 to 13,17 α-substituted 11 beta-halogen steroide, wherein R ⁷Represent CH ₃

15, as described 7 α of one of claim 11 to 14,17 α-substituted 11 beta-halogen steroide, wherein R ¹¹Represent fluorine.

16, as described 7 α of one of claim 11 to 15,17 α-substituted 11 beta-halogen steroide, wherein R ¹³Represent CH ₃

17, as described 7 α of one of claim 11 to 16,17 α-substituted 11 beta-halogen steroide, wherein R ¹⁷Represent H, CH ₃, C ₁-C ₁₈Alkynyl, CH ₂CN or CF ₃

18, as described 7 α of one of claim 11 to 17,17 α-substituted 11 beta-halogen steroide, wherein R ¹⁷It is ethynyl.

19, as described 7 α of one of claim 11 to 18,17 α-substituted 11 beta-halogen steroide, wherein R ¹⁷' represent H.

20, as described 7 α of one of claim 11 to 19,17 α-substituted 11 beta-halogen steroides, it is:

17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl is female-4-alkene-3-ketone,

Female-5 (10)-alkene of 17 α-ethynyl-11 β-fluoro-17 beta-hydroxies-7 Alpha-Methyl-3-ketone or

17 α-ethynyl-11 β-fluoro-7 Alpha-Methyls are female-1,3,5-triolefin-3,17-isoallopregnane-3.

21,7 α of general formula 6-substituted 11 beta-halogens are female-1,3, additive salt, ester and the acid amides of 5 (10)-triolefins and their pharmacology consistency:

R ³Represent H, C ₁-C ₄Alkyl, C ₁-C ₄Alkyloyl or have OR ³The C of the O atom of base ₃-C ₇Cyclic ethers,

R ⁷Be the P-Q base, and P is C ₁-C ₄Alkylidene group, Q are hydrogen, C ₁-C ₄Alkyl or C ₁-C ₄Fluoro-alkyl, and the P-Q base is bonded on the steroid backbone by P,

R ¹¹Be halogen,

R ¹³Be methyl or ethyl.

22,7 α as claimed in claim 21-substituted 11 beta-halogens are female-1,3,5 (10)-triolefins, and it is that 11 β-fluoro-3-hydroxyl-7 Alpha-Methyl is female-1,3,5 (10)-triolefins-17-ketone.

23, preparation is as 7 α of the described general formula 10 of one of claim 11 to 20, the method for 17 α-substituted 11 beta-halogen steroides, and wherein U-V-W-X-Y-Z represents ring structure C ¹-C ²-C ³-C ⁴=C ⁵-C ¹⁰, preparation method's step is as follows:

-use halo to remove hydroxylation reagent mutual-through type 4, nucleophilic substitution is carried out in the 11-position of 7 α of B-substituted 11 Alpha-hydroxy steroides;

-7 α-substituted 11 beta-halogen steroides that will so make and alkylating agent are optionally at the C of ring skeleton ¹⁷Atom reacts, and generates 7 α of general formula 10,17 α-substituted 11 beta-halogen steroides.

24, preparation is as 7 α of general formula 12 as described in one of claims 11 to 20, the method for 17 α-substituted 11 beta-halogen steroides, and wherein U-V-W-X-Y-Z represents ring structure C ¹-C ²-C ³-C ⁴-C ⁵=C ¹⁰, its method steps is as follows:

-use halo to remove hydroxylation reagent mutual-through type 4, nucleophilic substitution is carried out in the 11-position of 7 α of B-substituted 11 Alpha-hydroxy steroides;

-7 α-substituted 11 beta-halogen steroides that will so make and alkylating agent are optionally at the C of ring skeleton ¹⁷Atom reacts, and generates 7 α of general formula 10,17 α-substituted 11 beta-halogen steroides;

7 α of-isomerization general formula 10,17 α-substituted 11 beta-halogen steroides generate the isomer of corresponding general formula 12, and wherein U-V-W-X-Y-Z represents ring structure C ¹-C ²-C ³-C ⁴-C ⁵=C ¹⁰

25, preparation is as 7 α of one of claims 11 to 20 described general formula 8, the method for 17 α-substituted 11 beta-halogen steroides, and wherein U-V-W-X-Y-Z represents ring structure C ¹=C ²-C ³=C ⁴-C ⁵=C ⁶, its method steps is as follows:

-use halo to remove hydroxylation reagent mutual-through type 4, nucleophilic substitution is carried out in the 11-position of 7 α of B-substituted 11 Alpha-hydroxy steroides;

-7 α-substituted 11 beta-halogen steroide the oxidations that will so make, it is female-1,3 to generate 7 α as claim 17 or 18 described general formulas 6-substituted, 5 (10)-triolefins;

-7 α-substituted that will so make are female-1,3, and 5 (10)-triolefins and alkylating agent are optionally at the C of ring skeleton ¹⁷Atom reacts, and generates 7 α of general formula 8,17 α-substituted 11 beta-halogen steroides.

26, as 7 α of the described general formula 8,10,12 of one of claims 11 to 20, the application of 17 α-substituted 11 beta-halogen steroides in the preparation medicine.

27, a kind of pharmaceutical preparation, its contain at least a as claim 11 to 20 it-7 α of described general formula 8,10,12,17 α-substituted 11 beta-halogen steroides and at least a pharmacology consistency carrier.