KR20050026507A - Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids - Google Patents

Abstract

Disclosed is a new synthetic pathway for the production of precursors for the production of compounds having general formula (8,10,12). During said synthesis, compounds of general formal (4,B) are produced in a microbiological reaction. The meanings of radicals R7, R10, R11, R13, R 17 and R17' and group U-V-W-X-Y-Z are defined in the patent claims.

Description

7 Alpha-Substituted 11 Microbiological Methods for the Production of Alpha-hydroxy Steroids {MICROBIOLOGICAL METHOD FOR THE PRODUCTION OF 7 ALPHA-SUBSTITUTED 11 ALPHA-HYDROXYSTEROIDS}

The present invention relates to microbiological methods for the production of 7α-substituted 11α-hydroxy steroids, 7α, 17α-substituted 11β-halogen steroids that can be produced therefrom, methods of production for the compounds and their uses and containing these compounds A pharmaceutical formulation. The invention also relates to other 7α-substituted 11β-halogen steroids, namely 7α-substituted estra-1,3,5 (10) -triene, obtainable from 7α-substituted 11α-hydroxy steroids.

Androgens, in particular testosterone, are used for the treatment of menopause and for the development of male sex organs and for regulating male fertility. The hormone also has a partial anabolic active ingredient that promotes muscle growth.

Male menopause is characterized by an age-related decrease in endogenous androgen production, so that hormone replacement is performed for its treatment (HRT: hormone replacement therapy).

In addition to reducing spermatogenesis, administration of LH-RH for regulating male fertility results in release of LH and a decrease in testosterone levels and libido, which is compensated by administering testosterone preparations (DE 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 therapies using the administration of androgen and gestagenic active ingredients can be used for the regulation of male fertility (see for example WO 01/60376 A and the literature cited therein).

In the case of testosterone treatment, prostate hypertrophy (BPH: benign prostatic hyperplasia) has been shown to occur due to side effects, in particular due to an increase in the number of cells and the gland of the substrate. In the metabolism of testosterone mediated by 5α-reductase, dihydrotestosterone (DHT) is produced that can lead to the generation of BPH (Cummings et al., Ibid .; WO 99/13883 A1). Therefore, inhibition of 5α-reductase is used clinically to treat BPH (pinasteride).

Rapid metabolism of androgen steroid testosterone in the human body not only forms undesirable DHT, but also requires high doses of oral administration to reach the desired level of effect of testosterone. Thus, there is a need for alternative forms for administration, such as intramuscular injection or large patches.

In order to replace testosterone in the above-mentioned fields, 7α-methyl-19-nortestosterone (MeNT) has been proposed, which has a high binding affinity for androgen receptors and on the one hand has a high biological effectiveness such as testosterone. On the other hand, it is believed to resist metabolism by 5α-reductase due to steric hindrance by 7α-methyl groups (Cummings et al., Ibid., WO 99/13883 A1, WO 99/13812 A1, US-A-5,342,834 ).

During the metabolism of testosterone, some of these compounds are also reacted by the aromatization of ring A of the steroid system, particularly in the brain, liver and adipose tissue to form estradiol. With respect to the total action of testosterone and its metabolites, estradiol is substantially responsible for sex-specific behavior and gonadotropin regulation. Therefore, an action such as that of testosterone on adult males appears to be advantageous (Cummings et al., Ibid.).

However, the pharmacokinetic properties of testosterone have been found to be unsatisfactory. Especially in the case of oral administration, testosterone is rapidly excreted, resulting in unsatisfactory efficacy and duration of action of the produced pharmaceutical agent. Thus, other testosterone derivatives were synthesized. Such derivatives are described in US Pat. No. 5,952,319, in particular the 7α-, 11β-dimethyl derivatives of 19-nortestosterone, ie 7α, 11β-dimethyl-17β-hydroxyestr-4-en-3-one, 7α, 11β-dimethyl-17β-heptanoyloxyestr-4-en-3-one, 7α, 11β-dimethyl-17β-[[(2-cyclopentylethyl) -carbonyl] -oxy] -ester- 4-en-3-one, 7α, 11β-dimethyl-17β- (phenylacetyloxy) -estr-4-en-3-one, and 7α, 11β-dimethyl-17β-[[(trans-4- [ n-butyl] cyclohexyl) -carbonyl] -oxy] -estr-4-en-3-one.

The 7α, 11β-dimethyl derivatives have the same advantages as MeNT, including improved pharmacokinetic properties, ie their efficacy and time of action are improved over testosterone. However, these derivatives can only be produced via expensive synthetic methods.

The synthesis of steroids by microbiological methods is described in EP 0 900 283 B1. These include the group of Estre-4-en-3,17-dione and canrenone , including species of Aspergillus nigricans , Rhizopus arrhizus and Pestelotia . Microorganisms selected from can be converted to the corresponding 11α-hydroxy analogues. However, at the beginning of the description, Shibahara et al., Biochim. Biophys. Acta, 202 (1970), 172-179, reported that the microbiological 11α-hydroxylation reaction in steroids may be unpredictable.

The problem underlying the present invention is therefore to find derivatives of testosterone that are not sensitive to reduction with 5α-reductase and have improved pharmacokinetic properties and are particularly easy to produce. A very important aspect of the present invention is to find a way to have better access to the initial product, resulting in an easier initial product to be produced.

The problem on which the present invention is based is that the method for microbiological production of 7α-substituted steroids according to claims 1, 3 and 6, 7α, 17α-substituted 11β-halogen steroids according to claim 11, according to claims 23, 24 and 25 A method of producing a 7α, 17α-substituted 11β-halogen steroid, the use of a 7α, 17α-substituted 11β-halogen steroid according to claim 26, a pharmaceutical formulation containing a 7α, 17α-substituted 11β-halogen steroid according to claim 27, And 7α-substituted 11β-haloestra-1,3,5 (10) -triene according to claim 21. Preferred embodiments of the claimed subject matter are described in the dependent claims.

Justice

The following definitions relate to the specification, the entire part of the claims and the appended figure I.

All groups, radicals or other structural units may in each case vary independently of each other within the scope of the stated meaning.

All alkyl, alkylene, alkenyl, alkenylene, alkynyl, and alkynylene groups can be straight or branched chain. For example, the propenyl group can be described by one of the following chemical structures: -CH = C-CH ₃ , -CH ₂ -C = CH ₂ , or -C (CH ₃ ) = CH ₂ . Thus, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, neo-pentyl, 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 C Belongs to _1- to C ₁₈ -alkyl.

Alicyclic alkyl is cycloalkyl substituted with a cycloalkyl ring or one alkyl group or several alkyl groups bonded directly through a cycloalkyl ring or through one of the alkyl groups.

In the same way, alicyclic alkenyl is directly bonded through a cycloalkenyl ring or through one of an alkenyl group or optionally an alkyl group such that one or more double bonds are cycloalkenyl or one or more alkenyl contained in the alicyclic alkenyl. Cycloalkenyl or cycloalkyl substituted with one or more alkynyl and alkyl groups or with one or more alkyl groups.

Aryl, on the other hand, may be phenyl as well as 1-naphthyl or 2-naphthyl. In principle, aryl is also heteroaryl, in particular 2-, 3- and 4-pyridinyl, 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 acceptable addition salts are salts of inorganic or organic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, citric acid, oxalic acid, tartaric acid and methanesulfonic acid. The ester can be formed in particular with succinic acid.

The superscript numbers on the symbol R, for example R ¹³ , indicate their positions on the steroid ring skeleton, wherein the C atoms in the steroid ring skeleton are numbered according to the IUPAC nomenclature. The superscript number on symbol C, for example C ¹⁰ , indicates the position of each carbon atom on the steroid ring skeleton.

The novel microbiological process is used for the production of 7α-substituted 11α-hydroxy steroids of Formula 4B.

Where

R ⁷ is a PQ group, where

P represents C ₁ -to C ₄ -alkylene, Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl (partially or fully fluorinated alkyl) and the PQ group Is bound to the steroid skeleton via P,

R ¹⁰ means H, CH ₃ or CF ₃ ,

R ¹³ is methyl or ethyl.

In a first modified method for the production of this substance, suitable 7α-substituted steroids of Formula 3A were used as Aspergillus sp. , Beauveria sp. , Glomerella sp. , Nomo. California species (Gnomonia sp.), and the flow spokes Pasteurella species (Haplosporella sp.), and Rhizopus species crispus (Rhizopus sp.), using a microorganism selected from the group causes hydroxylation and oxidation in one process step comprising a.

Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as specified for the compound of formula 4B.

Particularly preferred are Aspergillus awamori , Aspergillus fischeri , Aspergillus malignus , Aspergillus niger , Beauveria bassiana , Glomerella cingulata , Nomonia cingulata , Haplosporella hesperedica and Rhizopus stolonifer , in particular Aspergillus awamori (CBS) ), Aspergillus Fischeri (ATCC 1020), Aspergillus Malignus (IMI 16061), Aspergillus niger (ATCC 9142), Biberia Bassiana (ATCC 7159), Glomerella Singulata (CBS 15226, CBS 23849, CBS 98069, ATCC 56596, ATCC 64682, IFO 6425), Nomonia Singgulata (CBS 15226), Haflosporella Hesperedica (CBS 20837) and Rizopus Stalloniper (ATCC 15441) are used.

Alternatively, this microbiological production method may be carried out in two stages where the hydroxylation reaction and the oxidation reaction occur in sequential reaction steps. The path of the reaction can be regulated through the reaction period: for example, by blocking the reaction after a certain reaction time, the hydroxylated but not yet oxidized species can be separated. Thus, two process steps can be performed separately or in mixed culture.

For this purpose, the compounds include Aspergillus spp., Viberia spp . , Gibberella sp. , Glomerella spp. , Nomonia spp. , Metarrhizium sp. , Nigrospora spp. sp. ), Rizopus spp. and Verticillium sp. spp . may be hydroxylated at the 11-position of Formula 3A in a first microbiological process step using a first microorganism selected from the group comprising: This results in the formation of 7α-substituted steroids having hydroxyl groups at the 11α-position. This compound has formula (C):

Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as described above for the compound of formula 4B. In particular, Aspergillus malignus , Aspergillus melleus , Aspergillus niger , Aspergillus ochraceus , Viveraa bassiana ), Gibberella fujikuroi , Gibberella zeae , Glomerella cingulata , Glomerella fusaroides , Gononia cingulata , Metarrhizium anisopliae , Nigrospora sphaerica , Rhizopus oryzae , Rhizopus stolonifer and Verticillium dahliae Is used. In this regard, in particular Aspergillus Malignus (IMI 16061), Aspergillus meleus (CBS), Aspergillus niger (ATCC 11394), Aspergillus okraceus (NRRL 405, CBS 13252, ATCC 46504) , Biberia Bassiana (ATCC 7159, IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS 12736), Gibberella Fuzikuroi (ATCC 14842), Gibberella Zeae (CBS 4474), Glomerella Singgulata (ATCC) 10534, CBS 23849, CBS 23749, ATCC 16646, ATCC 16052, IFO 6459, IFO 6425, IFO 6470, CBS 98069, IFO 7478, IFO 5257, ATCC 64682, ATCC 15470), Glomerella Fusaroides (ATCC 9552), Nomonia Singgulata (CBS 15226), Metadium Anisopliia (IFO 5940), Nigrospora Spa Erica (ATCC 12772), Rizopus Duck (ATCC 4858, ATCC 34102, ATCC 34102), Rizopus Stolo Nippers (ATCC 6227b, ATCC 15441) and Vertilium Dalea (ATCC 11405) are used for hydroxylation.

The intermediate C was then selected from the group comprising Bacillus sp. , Mycobacterium sp. , Nocardia sp. , And Pseudomonas sp. Oxidation is carried out in a second microbial processing step using microorganisms to form 7α-substituted 11α-hydroxy steroids of Formula 4B. Particularly Bacillus Rock Timor booth (Bacillus lactimorbus), Bacillus spa Erie Syracuse (Bacillus sphaericus), Mycobacterium neo Augusta Room (Mycobacterium neoaurum), Mycobacterium Smeg Matisse (Mycobacterium smegmatis), no-carboxylic Dia Coral Lena (Nocardia corallina , Nocardia globerula , Nocardia minima , Nocardia restrictus , Nocardia rubropertincta , Nocardia live monitor color (Nocardia salmonicolor) and Pseudomonas Testo Stephen Ronnie (Pseudomonas testosteroni) the use, in particular Bacillus Rock Timor booth (ATCC 245), Bacillus spa Erie Syracuse (ATCC 7055), Mycobacterium neo Augusta Room (ATCC 9626, NRRL B-3683, NRRL B-3805), Mycobacterium smegmatis (ATCC 14468), Nocardia Coralina (ATCC 31338), Nocardia Globeberula (ATCC 9356), Nocardi Minima (ATCC 19150), Nocardia Restrictus (NCIB 10027), Nocardia Lubropattinkt (ATCC 14352), Nocardia Salmonicolor (ATCC 19149) and Pseudomonas Testosterone (ATCC 11996) Used.

In another variation, the microbiological reaction can produce the compound of formula 4B from the 7α-substituted steroid of formula D:

Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as specified for the compound of formula 4B. The reactions include Aspergillus spp . , Viberia spp. , Curvularia sp. , Gibberella spp. , Glomerella spp. , Nomonia spp. , Haflosporella spp. , Helicostylum sp. ), Nigrospora species, lycopus species and Syncephalastrum sp. , Using a microorganism selected from the group wherein the steroid backbone is hydroxylated at the 11α-position to form a 7α- Substituted 11α-hydroxy steroids are generated. Aspergillus alliaceus , Aspergillus awamori , Aspergillus fischeri , Aspergillus malignus , Aspergillus melleus , Aspergillus nidualans , Aspergillus niger , Aspergillus ochraceus , Aspergillus variecolor , Aspergillus variecolor , Beauveria bassiana ), Curvularia lunata , Gibberella zeae , Glomerella cingulata , Glomerella fusaroides , Gnomonia cingulata , Haplosporella hesperedica , Helicostylum piriformae , nigrospora spaerica ( Nigrospora sphaerica ), Rhizopus oryzae and Syncephalastrum racemosum are preferred, especially Aspergillus aliaseus (ATCC 10060), Aspergillus awamori (CBS), Aspergillus Fischer (ATCC 1020), Aspergillus Marignus (IMI 16061), Aspergillus Meleus (CBS), Aspergillus nidualans (ATCC 11267), Aspergillus niger (ATCC 9142, ATCC 11394), Aspergillus okraceus (NRRL 405, ATCC 13252, ATCC 46504), Aspergillus varicolor (ATCC 10067), Biberia Bassiana (IFO 5838, ATCC 13144, IFO 4848, CBS 11025, CBS 12736, ATCC 7159) ), Kurbularia Lunata (IX3), Giberella Zeae (CBS 4474), Glomerella Singgulata (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), Nomonia Singgulata (CBS) 15226), haflosporella hesperadica (CBS 20837), helicostilum pyriforma (ATCC 8992), nigrospora spaerica (ATCC 12772), lycopus duck (ATCC 4858) and syncephalastrum Lasse Mow (IFO 4827) is used.

Particularly suitable are the processes in which the 7α-substituted 11α-hydroxy steroids of formula 4B are produced in which R ⁷ is CH ₃ and R ¹⁰ is H and R ¹³ is CH ₃ .

The method is performed in the usual way. For this purpose, a sterile nutrient solution is usually produced for the strain, and the strain is cultivated by inoculating the nutrient solution with the culture solution of the strain. The preculture produced in this way is placed in a fermentor optionally coated with a suitable elixir solution. Preferably, after the growth phase for the cultivation of the strain, the starting material (in this case, the compound of formula 3A or compound of formula D) may be added to the fermentor to proceed with the reaction according to the invention. After the reaction is complete, the mixture of substances is purified in a conventional manner to isolate the desired 7α-substituted 11α-hydroxy steroids.

From the compounds of formula 4B thus obtained, other compounds according to the invention can also be synthesized using the production process according to the invention. In particular, the 7α, 17α-substituted 11β-halogen steroids of Formulas 8, 10, 12 and their pharmaceutically acceptable addition salts, esters and amides produce beneficial active ingredients.

[Formula 8, 10, 12]

Where

UVWXYZ is C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ , C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ , or C ¹ -C ² -C ³ -C ⁴ One of the -C ⁵ -C ¹⁰ ring structures, in which case an oxo group (= O) is bonded to W (= C ³ ) or C ¹ = C ² -C ³ = C ⁴ -C ⁵ = C ⁶ ring Refers to the structure, in which case OR ³ radicals are bonded to W (= C ³ ),

R ³ means cyclic C ₃ -to C ₇ -ethers with O-atoms of H, C ₁ -to C ₄ -alkyl, C ₁ -to C ₄ -alkanoyl or OR ³ radicals,

R ⁷ means PQ group, where

P represents C ₁ -to C ₄ -alkylene, Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl (partially or fully fluorinated alkyl) and the PQ group Is bound to the steroid skeleton via P,

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

R ¹¹ is halogen,

R ¹³ is methyl or ethyl,

R ¹⁷ is H, C ₁ -to C ₁₈ -alkyl, alicyclic C ₁ -to C ₁₈ -alkyl, C ₁ -to C ₁₈ -alkenyl, alicyclic C ₁ -to C ₁₈ -alkenyl, C _1- to C ₁₈ -alkynyl, C ₁ to C ₁₈ -alkylaryl, C ₁ to C ₈ -alkylenenitrile or PQ group (PQ group has the above meaning),

R ^{17 '} is H, C ₁ -to C ₁₈ -alkyl, alicyclic C ₁ -to C ₁₈ -alkyl, C ₁ -to C ₁₈ -alkenyl, alicyclic C ₁ -to C ₁₈ -alkenyl, C ₁ - to C ₁₈ - alkynyl, or C ₁ - to C ₁₈ - alkyl and aryl, wherein R ^{17 'may} be coupled through a clay cake 17β- oxy groups, wherein R ^17' has one or more NR ¹⁸ R ¹⁹ Group or one or more SO _x R ²⁰ groups, where x is 0, 1 or 2, and R ¹⁸ , R ¹⁹ and R ²⁰ may each independently have the same meaning as R ¹⁷ have.

Obtained by further processing steps from the 7α-substituted 11α-hydroxy steroids of Formula 4B, these compounds are valuable active ingredients with strong androgen activity without the aforementioned side effects. These compounds are suitable for the production of active ingredients for pharmaceutical agents, in particular effective contraceptives and hormone replacement therapy (HRT).

In addition, if R ^{17 '} is substituted with an NR ¹⁸ R ¹⁹ group, it is a methylamino, dimethylamino, ethylamino, diethylamino, cyclohexylamino, dicyclohexylamino, phenylamino, diphenylamino, benzylamino or dibenzylamino group Can be.

Particularly suitable 7α, 17α-substituted 11β-halogen steroids of formulas 8, 10 and 12 have UVWXYZ of C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ , C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ or C ¹ = C ² -C ³ = C ⁴ -C ⁵ = C ¹⁰ A compound meaning a ring structure.

In the first case (when UVWXYZ is C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ ), these are steroids of formula 10:

In the second case (when UVWXYZ is C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ ), these are steroids of formula 12:

Compounds of formulas 10 and 12 are androgenic compounds.

In the third case (when UVWXYZ is C ¹ = C ² -C ³ = C ⁴ -C ⁵ = C ⁶ ), these are steroids of formula 8:

These compounds are estrogens (estrogen receptor-affinity compounds).

In all three cases, the radicals R ³ , R ⁷ , R ¹⁰ , R ¹¹ , R ¹³ , R ¹⁷ and R ^{17 ′} have the same meaning as the corresponding radicals in formulas 8, 10 and 12.

Independently of each other, R ¹ is preferably H and R ⁷ is CH ₃ and R ¹¹ is fluorine and R ¹³ is CH ₃ and R ¹⁷ is H, CH ₃ , C _1- to C ₁₈ -alkynyl, in particular ethynyl, CH ₂ CN or CF ₃ and / or R ^{17 ′} is H.

Particularly suitable according to the invention are the 7α, 17α-substituted 11β-halogen steroids of formula 8, 10, 12:

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).

For the production of this compound, the following production methods can be adopted:

For the production of 7α, 17α-substituted 11β-halogen steroids of formula 10, wherein UVWXYZ is C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ ring structure, the formula obtained by the microbiological production method according to the present invention 4B's 7α-substituted 11α-hydroxy steroids are used as starting material.

In the first synthetic step, the 7α-substituted 11α-hydroxy steroids thus obtained are converted to the corresponding 7α-substituted 11β-halogen steroids (5) by nucleophilic substitution using a halotalhydroxylation agent:

As the halogenohydroxylating agent, all compounds conventionally used for this purpose, for example hydrofluoric acid, hydrochloric acid, hydrobromic acid or hydroiodic acid, thionyl chloride or thionyl bromide, phosphorus pentachloride, phosphorus oxychloride, N-chloro Succinimide, triphenylphosphine / carbon tetrachloride, HF / pyridine or diethylaminosulfur trifluoride or preferably nonnapyl fluoride / 1,5-diazabicyclo [5.4.0] undecene are suitable.

Compound 10 is then produced from 5 by selective alkylation on C ¹⁷ of the ring backbone (see FIG. 1 in this regard). For selective alkylation, conventional alkylation reagents such as Grignard compounds and organometallic compounds, in particular alkyllithium compounds can be used. For example, ethynyl magnesium bromide as alkylating agent for the production of the corresponding 17α-ethynyl-17β-hydroxy-estr-4-en-3-one from ester-4-ene-3,17-dione Can be used.

For the production of 7α, 17α-substituted 11β-halogen steroids of Formula 12, wherein UVWXYZ is C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ ring structure, isomerization is carried out using the compound of Formula 10, The Δ ⁴ -double bond isomerized to Δ ^{5 (10)} -double bond. To protect the 3-keto group, first form a cyclic ether at position 3. Thereafter, the Δ ⁴ -double bond isomerizes to Δ ^{5 (10)} -double bond to form a 7α, 17α-substituted 11β-halogen steroid of Formula 12 and cleave the protecting group again.

The following method is used for the production of additional 7α, 17α-substituted 11β-halogen steroids of Formula 8 wherein the UVWXYZ is C ¹ = C ² -C ³ = C ⁴ -C ⁵ = C ¹⁰ :

First, as described above, the corresponding 11β-halogen steroid of formula 5 was subjected to halotalhydroxylation by nucleophilic substitution reaction from the 7α-substituted 11α-hydroxy steroid of formula 4B obtained by microbiological hydroxylation and oxidation. By forming.

Thereby, the 7α-substituted estra-1,3,5 (10) -triene of formula (6) is formed by oxidation with, for example, a copper (II) salt:

Wherein R ³ , R ⁷ , R ¹¹ and R ¹³ have the same meanings as described above. If R ³ is H, this compound can be synthesized directly. If it is another radical for R ³ , then the 1,3,5 (10) -triene ring must be formed by oxidation and then the corresponding ether or ester must be formed in a known manner.

The 7α-substituted 11β-haloestra-1,3,5 (10) -trienes of formula 6 and pharmaceutically acceptable addition salts, esters and amines thereof are also novel and therefore also according to the invention , 17α-substituted 11β-halogen steroids are claimed as intermediate products.

Particularly preferred 7α-substituted 11β-haloestra-1,3,5 (10) -triene of formula 6 is 11β-fluoro-3-hydroxy-7α-methylestra-1,3,5 (10) -tree Yen-17-on.

The 7α, 17α-substituted 11β-halogen steroids of Formula 8 according to the present invention are the same as described above for the synthesis of the compounds of Formula 10, the 7α-substituted 11β-haloestra-1,3,5 (10) From trienes by selective alkylation of the ring backbone at C ¹⁷ .

In addition, the 7α-substituted 11β-halogen steroid of Formula 9 may also be produced from a substance of Formula 4B obtained by microbiological hydroxylation and oxidation from a 7α-substituted steroid of Formula 3A or D, wherein the 7α-substituted 11β- Halogen steroids also have androgen action:

Wherein R ⁷ , R ¹¹ and R ¹³ have the same meanings as above. Particularly preferred compounds are 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one. Compounds of formula 9 and their pharmaceutically acceptable addition salts, esters and amides also have androgen action.

For the production of the compound of formula 9, ester-4-en-3,17-dione (5) can be converted to 17β-hydroxy-estr-4-en-3-one (9) using, for example, boron hydride. Reduction).

In addition, compounds of formula 9 may be converted to the corresponding 7α-substituted 11β-haloestra-5 (10) -ene:

Wherein R ⁷ , R ¹⁰ , R ^11, and R ¹³ have the same meanings as in Formulas 8, 10, and 12. For this purpose, the compound of formula 9 isomerized by changing the Δ ⁴ -double bond to Δ ^{5 (10)} -double bond. To protect the 3-keto group, first form a cyclic ether in the 3-position. The Δ ⁴ -double bond is then isomerized to Δ ^{5 (10)} -double bond to form the 7α-substituted 11β-halogen steroid, after which the protecting group is cleaved again.

Finally, the corresponding 7α-substituted 11β-haloestra-5 (10) -ene can be converted from the compound of formula 5 by isomerizing Δ ⁴ -double bonds to Δ ^{5 (10)} -double bonds:

Wherein R ⁷ , R ¹⁰ , R ^11, and R ¹³ have the same meanings as in Formulas 8, 10, and 12. For this purpose, the compound of formula 5 is isomerized by changing the Δ ⁴ -double bond to Δ ^{5 (10)} -double bond. To protect the 3-keto group, first form a cyclic ether in the 3-position. The Δ ⁴ -double bond is then isomerized to Δ ^{5 (10)} -double bond to form the 7α-substituted 11β-halogen steroid, and the protecting group is cleaved again.

All of the above compounds can also be further esterified or etherified if the corresponding hydroxyl group is present at the 3- or 17β-position. For example, compound (9) can be converted to the corresponding 17β-ether or 17β-ester. Preferred compounds are 11β-fluoro-17β- (4-sulfaylbenzoxoxy) -7α-methylestr-4-en-3-one. As substituents on the oxy-oxygen atoms in C ¹⁷ , basically the same radicals as mentioned for R ^{17 ′} are suitable.

In particular, 7α, 17α-substituted 11β-halogen steroids of Formulas 8, 10, 12 are suitable for the production of pharmaceutical agents. Accordingly, the present invention also provides the use of a compound of formula 8, 10, 12 for the production of a pharmaceutical formulation and a pharmaceutical formulation containing at least one compound of formula 8, 10, 12 and at least one pharmaceutically acceptable carrier. It is about.

The 7α, 17α-substituted 11β-halogen steroids of formulas 10 and 12 according to the invention are compounds with strong androgenic action without the above mentioned side effects such as, for example, stimulation of the prostate (especially do not cause benign prostatic hyperplasia). This compound is easy to synthesize. The compounds according to the invention of formula (10) or (12) can be used for male HRT as well as other active ingredients provided that they have sufficient dose control to reduce blood levels of LH, testosterone and FSH (follicle stimulating hormone) produced in the body. Also suitable as adequately effective male contraceptives without additional administration. This depends on the 11β-halogen steroid according to the invention to inhibit the release of LH and FSH. LH stimulates Radig cells to secrete testosterone. If the blood levels of LH remain low, the secretion of endogenous testosterone is also reduced. Testosterone is required for spermatogenesis, and FSH stimulates germ cells. Thus, sufficiently high blood levels of FSH and LH are required for effective spermatogenesis, and sufficiently high LH blood levels result in testosterone release, which is required for spermatogenesis.

Since treatment with 7α, 17α-substituted 11β-halogen steroids alone can lead to effective male contraception without additional active ingredients for infertility, the administration of pharmaceutical agents suitable for this purpose can be significantly simplified and the cost of use significantly reduced. Can be.

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

In addition, the 7α, 17α-substituted 11β-halogen steroids according to the present invention are 5α-reductase and steroid-11-hydroxylase [CYP11B (P450c11), G. Zhang, WL Miller, Journal of Clinical Endocrinology and Metabolism, Vol. . 81, pages 3254-3256 (1996), effectively inhibiting the prostate irritation, for example, and these compounds have improved pharmacokinetic properties. Inhibition of 11-hydroxylase reduces inactivation of androgenic compounds and reduces excretion from the human body. As a result, the effectiveness and duration of action of these compounds are improved, especially after oral administration, as compared to known compounds.

For this reason, these compounds have an aromatization ability to form estrogen steroids obtained simultaneously with a reduced tendency to 5α-reduction and have a beneficial effect on serum lipids and the central nervous system and in particular for use in regulating male fertility and Suitable for androgen replacement therapy.

The observation of androgen action and the absence of such side effects was determined by seminal vesicle tests on the compounds of formulas 10 and 12 according to the invention. The effectiveness of the compound of formula 8 according to the present invention was measured for estrogen activity by uterine growth test.

The 7α, 17α-substituted 11β-halogen steroids of formula 10 or 12 according to the invention or pharmaceutical preparations according to the invention containing these compounds are very suitable for the treatment of male males of fertility and basically male mammals. As a result of the application for male contraception male patients are only temporarily infertile. After the application of the active ingredient or pharmaceutical preparation according to the invention is complete, it is returned to its original state so that the male patient is no longer infertile, and sperm production again occurs to its original level. In order to maintain a temporary infertility for a desired period of time, the administration of the active ingredient or agent should be carried out continuously, depending on the form of administration, the administration should be repeated daily, at shorter intervals or at longer intervals do. After a single or repeated administration of the active ingredient or agent, the fertility of the male patient only recovers slowly rather than immediately, with the duration required for this purpose being dependent on several factors, such as the dose, the constitution of the patient and Rely on parallel administration of other pharmaceutical agents.

If the purpose of administration is contraception, the dose of 7α, 17α-substituted 11β-halogen steroids should have a blood level of LH and FSH of 2.5 IE / ml (IE: international units) or less in each case, in particular 1.0 IE / ml or less. Blood levels should be kept high, below 10 nmol / l, especially below 3 nmol / l.

If the 7α, 17α-substituted 11β-halogen steroids according to the invention are used in HRT without achieving the purpose of contraception, the dose remains lower. In this case, blood levels of LH and FSH are greater than 2.5 I.E./ml, respectively, and an attempt is made to achieve an effect value such that blood levels of testosterone can be greater than 10 nmol / l.

The dose of the 7α, 17α-substituted 11β-halogen steroids of formula 10 or 12 according to the invention required to maintain blood levels of LH, FSH and testosterone depends on many factors and should be determined in a dose-specific manner. First, the dose naturally depends on the type of therapy. If the compound is to be used for male contraception, it should be given a significantly higher dose than for use for HRT. The dose also depends on the type of 7α, 17α-substituted 11β-halogen steroids and their bioavailability. The type of administration is also essential for the dosage. Finally, the dose also depends on the condition of the patient to be treated and whether other factors are provided in parallel, for example other pharmaceutical agents.

Compounds are orally and parenterally, for example, i.p. (intraperitoneal), i.v. (intravenous), i.m. (Intramuscular) or transdermally. The compound may also be implanted into tissue. The amount of compound to be administered can be varied within a wide range if an effective amount is administered. Based on the condition to be treated and the type of dosage, the amount of compound administered can vary within wide ranges. In humans, the daily dose ranges from 0.1 to 100 mg. The preferred daily dose for humans is 0.1 to 10 mg. The duration of administration depends on the purpose to be achieved.

Capsules, pills, tablets, coated tablets, creams, ointments, lotions, solutions, for example syrups, gels, injections, for example i.p., i.v., i.m. Or transdermal injection or the like, and each dosage form releases the total amount of the compound according to the invention gradually or within a short time depending on its type.

For oral administration, capsules, pills, tablets, coated tablets and solutions or other known oral dosage forms are used as pharmaceutical preparations. In this case, the pharmaceutical formulation may be formulated in such a way that it releases the active ingredient in a short time and delivers it into the body or has a depot action so that the active ingredient is delayed and slowly supplied into the body. In addition to the 7α, 17α-substituted 11β-halogen steroids, the dosage unit may comprise one or more pharmaceutically acceptable carriers, such as substances, surfactants, solubilizers, microcapsules, microparticles, granules for adjusting the rheology of pharmaceutical agents. , Diluents, binders such as starch, sugars, sorbitol and gelatin, and also fillers such as silicic acid and talc, lubricants, dyes, flavors and other substances.

In particular, the 7α, 17α-substituted 11β-halogen steroids according to the present invention are for oral administration and may be formulated in the form of a solution containing the following components in addition to the active 11β-halogen steroid: pharmaceutically acceptable oils and / or Pharmaceutically acceptable lipophilic surfactants and / or pharmaceutically acceptable hydrophilic surfactants and / or pharmaceutically acceptable water-miscible solvents. In this regard, see WO-A-97 / 21440.

In order to obtain better bioavailability of the steroid, the compound may be formulated as a cyclodextrin clathrate. For this purpose the compound is reacted with α-, β-, γ-cyclodextrin or its derivatives.

If creams, ointments, lotions and liquids that can be applied topically are used, they should be so configured that the compounds according to the invention can be supplied in a sufficient amount in the body. In this form for administration, adjuvants, for example substances for adjusting the rheology of pharmaceutical agents, surfactants, preservatives, solubilizers, diluents, substances for increasing the permeability through the skin of the steroids according to the invention, dyes, Perfumes and skin protectants such as conditioners and moisturizers are included. In addition to the steroids according to the invention, other active ingredients may also be contained in pharmaceutical pharmaceuticals.

For parenteral administration, the active ingredient may be dissolved or suspended in a physiologically acceptable diluent. As diluents, oils with or without solubilizers, surfactants, suspending agents or emulsifiers are very frequently used. Examples of oils used are olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil. In order to prepare an injection preparation, any liquid carrier may be used in which the compound according to the invention is dissolved or emulsified. This liquid also often contains substances for controlling viscosity, surfactants, preservatives, solubilizers, diluents and other additives to keep the solution isotonic. Other active ingredients may also be administered with the 7α, 17α-substituted 11β-halogen steroids.

The 11β-halogen steroids according to the present invention may be administered, for example subcutaneously, in the form of depot injection or implantable preparations which may be formulated to allow delayed release of the active ingredient. For this purpose, known techniques can be used, for example depots which dissolve or act with the membrane. Implants may contain, for example, biodegradable polymers or inert materials such as synthetic silicones such as silicone rubber. The 11β-halogen steroids according to the present invention may also be introduced into the patch, for example for transdermal administration.

The following examples are used to illustrate the invention in more detail:

A. Microbiological Synthesis:

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

Example 1:

Sterilized in 121 ° C. autoclave for 30 minutes, 3 wt% glucose, 1 wt% corn steep liquor, 0.2 wt% NaNO ₃ , 0.1 wt% KH ₂ PO ₄ , 0.2 wt% K ₂ liter Ellenmeyer flask containing 1000 ml of nutrient solution consisting of ₂ HPO ₄ , 0.05 wt% KCl, 0.05 wt% MgSO ₄ · 7H ₂ O and 0.002 wt% FeSO ₄ · 7H ₂ O (pH 6.0) Was inoculated in a slope bar culture of Gnomonia cingulata (CBS 15226) strain and shaken at 165 rpm in a rotary shaker at 28 ° C. for 72 hours. This preculture was inoculated with a 20 liter incubator covered with 19 liters of sterile medium of the same final composition as described for the preculture. In addition, prior to sterilization, 1.0 ml of silicone oil and 1.0 ml of synperonic (oxoalcohol ethoxylate) were added for foam reduction. 4.0 g of 17β-hydroxy-7α-methylester-4 in 40 ml DMF after a growth period of 12 hours at an overpressure of 0.7 bar, a temperature of 28 ° C., aeration of 20 L / min and agitation speed of 250 rpm. A solution of -en-3-one was added. Stirring was continued and vented. After 135 hours, the culture broth was collected and extracted with 10 liters of methyl isobutyl ketone for 12 hours and with 5 liters of methyl isobutyl ketone for 5 hours. The combined organic phases were evaporated to dryness. The silicone oil was washed with hexane. After chromatography on silica gel with a gradient of hexane and ethyl acetate, 1.64 g (39%) of 11α-hydroxy-7α-methylestr-4-ene-3,17-dione was isolated.

Example 2

Sterilized in 121 ° C. autoclave for 30 minutes, 3 wt% glucose, 1 wt% corn leachate, 0.2 wt% NaNO ₃ , 0.1 wt% KH ₂ PO ₄ , 0.2 wt% K ₂ HPO ₄ , 0.05 A 2 liter Elenmeyer flask containing 1000 ml of nutrient solution consisting of wt.% KCl, 0.05 wt.% MgSO ₄ .7H ₂ O and 0.002 wt.% FeSO ₄ .7H ₂ O (pH 6.0) The inoculation was carried out in a four- bar rod culture of Glomerella cingulata (IFO 6425) strain and shaken at 165 rpm on a rotary shaker at 28 ° C. for 72 hours. This preculture was inoculated with a 20 liter fermenter, which was coated with 19 liters of sterile medium of the same final composition as described for the preculture. In addition, prior to sterilization, 1.0 ml of silicone oil and 1.0 ml of synferonics were added for foam reduction. 2.0 g of 17β-hydroxy-7α-methylester-4 in 30 ml DMF after a growth period of 12 hours at an overpressure of 0.7 bar, a temperature of 28 ° C., aeration of 10 L / min and a stirring speed of 350 rpm. A solution of -en-3-one was added. Stirring was continued and vented. After 19 hours, the culture broth was collected and extracted with 20 liters of methyl isobutyl ketone for 16 hours and extracted with 20 liters of methyl isobutyl ketone for 23 hours. The combined organic phases were evaporated to dryness. The residue was dissolved in large amount of methanol. Silicone oil was filtered off. It was concentrated by evaporation, chromatographed on silica gel with a gradient of dichloromethane and acetone, followed by 1.55 g (73%) of 11α, 17β-dihydroxy-7α-methylestr-4-en-3-one Was isolated. After recrystallization with acetone / diisopropyl ether, 827 mg (39%) of white crystals having a melting point of 163 ° C. and having a [α] _D = -16 ° (CHCl ₃ , c = 0.501) were isolated.

Sterilized in a 121 ° C. autoclave for 30 minutes, with 0.5 wt% glucose, 0.5 wt% bacto-yeast extract, 0.1 wt% peptone, and 0.2 wt% corn leach (pH 7.5) A 2 liter Ellenmeyer flask containing 500 ml of nutrient solution composed was inoculated with 4 cyrospheres from the culture of Bacillus sphaericus (ATCC 7055) strain and a 28 ° C. rotary shaker for 24 hours. At 165 rpm. In this preculture, four 2 liter Ellenmeyer flasks containing 500 ml of sterile medium of the same composition as described for the preculture were inoculated with 10% of each of these culture broths. 50 mg of 11α, 17β-dihydroxy-7α-methylestr-4-en-3-one in 2.5 ml of DMF after the growth phase in a rotary shaker at 165 rpm for 4 hours at a temperature of 28 ° C. The solution was added to each flask. Shaking 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 sulphate and evaporated to dryness. In this case, 630 mg of oily-crystalline residue were obtained. After recrystallization with acetone / diisopropyl ether, 103 mg (49.2%) of yellow crystals having a melting point of 189 ° C. and having a [α] _D = + 40.4 ° (CHCl ₃ , c = 0.529) were isolated (chromatography above). Direct crystallization without purification).

Example 3

Sterilized in a 121 ° C. autoclave for 30 minutes, 3% glucose, 1% corn leachate, 0.2% NaNO ₃ , 0.1% KH ₂ PO ₄ , 0.2% K ₂ HPO ₄ , 0.05 Aspergillus Okra, a 2 liter Ellenmeyer flask containing 500 ml of nutrient solution consisting of KCl by weight, 0.05% by weight MgSO ₄ .7H ₂ O and 0.002% by weight FeSO ₄ .7H ₂ O (pH 6.0) Half-slope rod cultures of the Aspergillus ochraceus strain were inoculated and shaken at 165 rpm on a rotary shaker at 28 ° C. for 72 hours. This preculture was inoculated with a 10 liter incubator, which was coated with 9.5 liters of sterile medium of the same final composition as described for the preculture. In addition, prior to sterilization, 0.5 ml of silicone oil and 0.5 ml of synferonics were added for foam reduction. 1.0 g of 7α-methylester-4-ene-3 in 15 ml DMF after a growth period of 6 hours at an overpressure of 0.7 bar, a temperature of 28 ° C., aeration of 5 L / min and a stirring speed of 350 rpm, A solution of 17-dione was added. Stirring was continued and vented. After 22 hours, the culture broth was collected and extracted with 7 liters of methyl isobutyl ketone for 4 hours. The combined organic phases were evaporated to dryness. The residue was dissolved in large amount of methanol. Silicone oil was filtered off. It was concentrated by evaporation and chromatographed on silica gel with a gradient of dichloromethane and acetone, followed by 0.78 g (74%) of 11α-hydroxy-7α-methylest-4-ene-3,17-dione. Isolated. After recrystallization with acetone / diisopropyl ether, 311 mg (29.6%) of white crystals having a melting point of 200 ° C. and having a [α] _D = + 52 ° (CHCl ₃ , c = 0.5905) were isolated.

B. Chemical Production Methods

Example 4 Production of 11β-Fluoro-17β-hydroxy-7α-methylestr-4-en-3-one

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

11.5 ml of perfluorobutane-1-sulfonic acid fluoride was added to 13.08 g of 11α- in 250 ml of toluene and 18.2 ml of 1,8-diazabicyclo [5,4,0] undec-7-ene. To a solution of hydroxy-7α-methyl-estr-4-ene-3,17-dione (produced by the microbiological synthesis according to the invention [Part A]) was added dropwise at 0 ° C. After 1 h, it was neutralized with 2 M hydrochloric acid, added to water, extracted four times with ethyl acetate, washed with saturated sodium chloride solution, dried and concentrated by evaporation under vacuum. The crude product was chromatographed on silica gel with a hexane / ethyl acetate gradient to afford 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 tetrahydrofuran was added at 1 ° C. in 1M lithium aluminum tri-tert-butoxy in tetrahydrofuran. Mix dropwise with 29.5 ml of hydride and stir at 0 ° C. for 5.5 h. Dilute sulfuric acid is then added at 0 ° C., the reaction solution is added to ice water, extracted three times with ethyl acetate, washed with neutral, dried over sodium sulfate, concentrated by evaporation under vacuum and concentrated with hexane / ethylacetate Chromatography on silica gel. 5.8 g of 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one having a melting point of 143-144 ° C was obtained. [a] _D = + 89.9 ° (CHCl ₃ ).

Example 5: Production of 11β-Fluoro-17β- (4-sulfaylbenzoxoxy) -7α-methylestr-4-en-3-one

A solution of 500 mg 11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one in 7.5 ml pyridine was added at room temperature to 750 mg 4-sulfamoylbenzoic acid, 800 mg N, It was mixed with N-dicyclohexylcarbodiimide and 125 mg of p-toluenesulfonic acid and stirred for 8.5 hours. Then it was added to sodium bicarbonate solution, extracted four times with dichloromethane, washed with neutral, dried over sodium sulfate and concentrated by evaporation under vacuum and chromatographed on silica gel with dichloromethane / acetone. 302 mg of 11β-fluoro-17β- (4-sulfaylbenzoxoxy) -7α-methylestr-4-en-3-one having a melting point of 232 ° C was obtained. [a] _D = + 100.5 ° (CHCl ₃ ).

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

a) 11β-fluoro-3-methoxy-7α-methylestra-3,5-diene-17-one:

A solution of 2 g of 11β-fluoro-7α-methylester-4-ene-3,17-dione in 20 ml of 2,2-dimethoxypropane was 200 mg of pyridinium tosylate at 80 ° C. for 6.5 hours. Stir with. Then diluted with ethyl acetate, washed with sodium bicarbonate solution and sodium chloride solution, dried over sodium sulfate and concentrated by evaporation under vacuum. 2 g of crude 11β-fluoro-3-methoxy-7α-methylestra-3,5-diene-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 74.2 ml of ethynylmagnesium bromide solution (0.5 M in tetrahydrofuran) at 0 ° C. and stirred at 0 ° C. for 1 hour. It was. Thereafter, a solution of 0.2 g of crude 11β-fluoro-3-methoxy-7α-methylestra-3,5-diene-17-one in 40 ml of tetrahydrofuran is added dropwise and for 3.5 hours at 0 ° C. Further stirred. To finish, saturated ammonium chloride solution is added, added to water, extracted three times with ethyl acetate, washed with semi-concentrated hydrochloric acid, sodium bicarbonate solution and sodium chloride solution, dried over sodium sulphate, concentrated by evaporation under vacuum Chromatography on silica gel with hexane / ethyl acetate. 1.15 g of pure 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one having a melting point of 218-220 ° C. was obtained. [a] _D = + 19.2 ° (CHCl ₃ ).

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

a) 3,3-ethanediyldioxy-17α-ethynyl-11β-fluoro-7α-methylestr-5 (10) -ene-17β-ol:

A solution of 700 mg of 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 2.3 ml of trimethyl Stir for 6.5 hours at room temperature with orthoformate and 30 mg of p-toluenesulfonic acid hydrate. Then it was added to sodium bicarbonate solution, extracted three times with ethyl acetate, washed neutrally, dried over sodium sulfate, concentrated by evaporation under vacuum, and chromatographed on silica gel with hexane / ethyl acetate. 205 mg of 3,3-ethanediyldioxy-17α-ethynyl-11β-fluoro-7α-methylester-5 (10) -ene-17β-ol were obtained.

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

Of 205 mg of 3,3-ethanediyldioxy-17α-ethynyl-11β-fluoro-7α-methyl-estr-5 (10) -ene-17β-ol in 27 ml methanol and 3.6 ml water The solution was stirred with 361 mg of oxalic acid at room temperature for 24 hours. Then it was added to sodium bicarbonate solution, extracted three times with ethyl acetate, washed neutrally, dried over sodium sulfate, concentrated by evaporation under vacuum and chromatographed on silica gel with hexane / ethyl acetate. 95 mg of 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-5 (10) -en-3-one having a melting point of 112-114 ° C was obtained.

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

a) 11β-fluoro-3-hydroxy-7α-methylestra-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 bromide (II) at 25 ° C. for 6.5 hours. . Then diluted with ethyl acetate, washed with sodium bicarbonate solution and sodium chloride solution, dried over sodium sulfate, evaporated to dryness in vacuo, and chromatographed on silica gel with hexane / acetone. 280 mg of pure 11β-fluoro-3-hydroxy-7α-methylestra-1,3,5 (10) -trien-17-one having a melting point of 185-186 ° C. was obtained.

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 is mixed dropwise with 16.5 ml of ethynylmagnesium bromide solution (0.5 M in tetrahydrofuran) at 0 ° C. and 0.5 hour at 0 ° C. Stirred. Then, 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 added dropwise, and 0 The mixture was further stirred for 3.5 h. To finish, saturated ammonium chloride solution was added, added to water, extracted four times with ethyl acetate, washed neutrally, dried over sodium sulfate, concentrated by evaporation in vacuo and chromatographed on silica gel with hexane / ethyl acetate. .

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

Claims (27)

The 7α-substituted steroid of Formula 3A was used as Aspergillus sp. , Beauveria sp. , Glomerella sp. , Gnomonia sp. , Haflosporella. species (Haplosporella sp.), and Rhizopus species crispus (Rhizopus sp.), using a microorganism selected from the group consisting of hydroxylation, and, microbiological production methods of 7α- substituted 11α- hydroxy-steroid of the formula 4B oxidizing. <Formula 4B> Where R ⁷ is a PQ group, where P represents C ₁ -to C ₄ -alkylene, Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl, the PQ group is bonded via P to the steroid backbone, R ¹⁰ may be in the α- or β-position and means H, CH ₃ or CF ₃ , R ¹³ is methyl or ethyl. <Formula 3A> Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as above. The microorganism of claim 1, wherein the microorganism is Aspergillus awamori , Aspergillus fischeri , Aspergillus malignus , Aspergillus niger , Aspergillus niger , Biberia bash Groups that include Beauveria bassiana , Glomerella cingulata , Gnomonia cingulata , Haplosporella hesperedica , and Rhizopus stolonifer The method selected from. The 7α-substituted steroid of Formula 3A was used as Aspergillus sp. , Beauveria sp. , Gibberella sp. , Glomerella sp. , Nomonia sp. Selected from the group comprising Gnomonia sp. ), Metarrhizium sp. , Nigrospora sp. , Rhizopus sp. , And Verticillium sp. 1 Microorganisms in the first microbiological process step using microorganisms to form 7α-substituted 11α-hydroxy steroids of Formula C, and produced 7α-substituted 11α-hydroxy steroids of Formula C. Bacillus sp .), Mycobacterium species (Mycobacterium sp.), no carboxylic dia species (Nocardia sp.), and Pseudomonas species (Pseudomonas sp.), in a second microbiological process step of using a second microorganism selected from the group comprising By oxidizing to 7α-substituted A method of microbiological production of a 7α-substituted 11α-hydroxy steroid of Formula 4B, which forms an Lloyd. <Formula 4B> Where R ⁷ is a PQ group, where P represents C ₁ -to C ₄ -alkylene and Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl, the PQ group is bonded via P to the steroid backbone, R ¹⁰ may be in the α- or β-position and means H, CH ₃ or CF ₃ , R ¹³ is methyl or ethyl. <Formula 3A> Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as above. <Formula C> Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as above. The method of claim 3, wherein the first microorganism is Aspergillus malignus , Aspergillus melleus , Aspergillus niger , Aspergillus nigerus Aspergillus ochraceus , Beauveria bassiana , Gibberella fujikuroi , Gibberella zeae , Glomerella cingulata , Glomerella fusaroides , Gnomonia cingulata , Metarrhizium anisopliae , Nigrospora sphaerica , Rhizopus oryzae , Rhizopus stolonifer and Verticillium dahliae . The method of claim 3 or claim 4, wherein the second microorganism is Bacillus lock Timor booth (Bacillus lactimorbus), Bacillus spa Erie kusu (Bacillus sphaericus), Mycobacterium neo brother room (Mycobacterium neoaurum), Mycobacterium Smeg Mycobacterium smegmatis , Nocardia corallina , Nocardia globerula , Nocardia minima , Nocardia restrictus , Nocara Dia Lube Roper tin Ketapang (Nocardia rubropertincta), the method no-carboxylic Dia flesh color monitor (Nocardia salmonicolor) and Pseudomonas Testo Ste Ronnie is selected from the group including (Pseudomonas testosteroni). The 7α-substituted steroids of Formula D were used as Aspergillus sp. , Beauveria sp. , Curvularia sp. , Gibberella sp. , Glomerella spp. (Glomerella sp.), Nomo California species (Gnomonia sp.), and the flow Spokane Pasteurella species (Haplosporella sp.), Halley's nose still room species (Helicostylum sp.), Negro, Castello La species (Nigrospora sp.), Rizzo crispus species A method for microbiological production of 7α-substituted 11α-hydroxy steroids of Formula 4B, which is hydroxylated with a microorganism selected from the group comprising Rhizopus sp. And Syncephalastrum sp . <Formula 4B> Where R ⁷ is a PQ group, where P represents C ₁ -to C ₄ -alkylene and Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl, the PQ group is bonded via P to the steroid backbone, R ¹⁰ means H, CH ₃ or CF ₃ , R ¹³ is methyl or ethyl. <Formula D> Wherein R ⁷ , R ¹⁰ and R ¹³ have the same meanings as above. The microorganism of claim 6, wherein the microorganism is Aspergillus alliaceus , Aspergillus awamori , Aspergillus fischeri , Aspergillus malignus , Aspergillus aiamori Aspergillus melleus , Aspergillus nidualans , Aspergillus niger , Aspergillus ochraceus , Aspergillus variecolor , Beauveria bassiana , Curvularia lunata , Gibberella zeae , Glomerella cingulata , Glomerella fusaroides , Nomo Gnomonia cingulata , Haplosporella hesperedica , Helicostylum piriformae , Nigrospora sphaerica , Rhizopus oryzae and Syncephalastrum racemosum . 8. The microbiological method of claim 1, wherein R ⁷ is CH _3. ⁹ . 9. The microbiological method of claim 1, wherein R ¹⁰ is H. ¹⁰ . The microbiological method of any one of claims 1-9, wherein R ¹³ is CH ₃ . 7α, 17α-substituted 11β-halogen steroids of Formulas 8, 10, 12 and pharmaceutically acceptable addition salts, esters and amides thereof. <Formula 8, 10, 12> Where UVWXYZ is C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ , C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ , or C ¹ -C ² -C ³ -C ⁴ One of the ring structures of —C ⁵ -C ¹⁰ , in which case an oxo group (═O) is bonded to W (= C ³ ) or C ¹ = C ² -C ³ = C ⁴ -C ⁵ = C ⁶ A ring structure, in which case OR ³ radicals are bonded to W (= C ³ ), R ³ means cyclic C ₃ -to C ₇ -ethers with O-atoms of H, C ₁ -to C ₄ -alkyl, C ₁ -to C ₄ -alkanoyl or OR ³ radicals, R ⁷ means PQ group, where P represents C ₁ -to C ₄ -alkylene and Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl, the PQ group is bonded via P to the steroid backbone, R ¹⁰ may be in the α- or β-position and represents H, CH ₃ or CF ₃ , and is present only when XYZ is not C ⁴ -C ⁵ = C ¹⁰ , R ¹¹ is halogen, R ¹³ is methyl or ethyl, R ¹⁷ is H, C ₁ -to C ₁₈ -alkyl, alicyclic C ₁ -to C ₁₈ -alkyl, C ₁ -to C ₁₈ -alkenyl, alicyclic C ₁ -to C ₁₈ -alkenyl, C _1- to C ₁₈ -alkynyl, C ₁ to C ₁₈ -alkylaryl, C ₁ to C ₈ -alkylenenitrile or PQ group (PQ group has the above meaning), R ^{17 '} is H, C ₁ -to C ₁₈ -alkyl, alicyclic C ₁ -to C ₁₈ -alkyl, C ₁ -to C ₁₈ -alkenyl, alicyclic C ₁ -to C ₁₈ -alkenyl, C ₁ - to C ₁₈ - alkynyl, or C ₁ - to C ₁₈ - alkyl and aryl, wherein R ^{17 'may} be coupled through a clay cake 17β- oxy groups, wherein R ^17' is added to one or more NR ¹⁸ May be substituted with a R ¹⁹ group or one or more SO _x R ²⁰ groups, where x is 0, 1 or 2, and R ¹⁸ , R ¹⁹ and R ²⁰ may each independently have the same meaning as R ¹⁷ have. The UVWXYZ of claim 11, wherein the UVWXYZ is C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ or C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ or C ¹ = C ^2- 7α, 17α-substituted 11β-halogen steroids with C ³ = C ⁴ -C ⁵ = C ¹⁰ ring structures. The 7α, 17α-substituted 11β-halogen steroid according to claim 11 or 12, wherein R ¹ is H. The 7α, 17α-substituted 11β-halogen steroid according to any one of claims 11 to 13, wherein R ⁷ is CH ₃ . The 7α, 17α-substituted 11β-halogen steroid according to any one of claims 11 to 14, wherein R ¹¹ is fluorine. The 7α, 17α-substituted 11β-halogen steroid according to any one of claims 11 to 15, wherein R ¹³ is CH ₃ . The 7α, 17α-substituted 11β-halogen steroid according to any one of claims 11 to 16, wherein R ¹⁷ is H, CH ₃ , C ₁ -to C ₁₈ -alkynyl, CH ₂ CN or CF ₃ . The 7α, 17α-substituted 11β-halogen steroid according to any one of claims 11 to 17, wherein R ¹⁷ is ethynyl. The 7α, 17α-substituted 11β-halogen steroid according to any one of claims 11 to 18, wherein R ^{17 '} is H. The method according to any one of claims 11 to 19, 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-4-en-3-one, 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methylestr-5 (10) -en-3-one, or 7α, 17α-substituted 11β-halogen steroid, which is 17α-ethynyl-11β-fluoro-7α-methylestra-1,3,5 (10) -triene-3,17β-diol.  7α-substituted 11β-haloestra-1,3,5 (10) -triene and a pharmaceutically acceptable addition salt, ester and amide thereof. <Formula 6> Where R ³ means cyclic C ₃ -to C ₇ -ethers with O-atoms of H, C ₁ -to C ₄ -alkyl, C ₁ -to C ₄ -alkanoyl or OR ³ radicals, R ⁷ is a PQ group, where P represents C ₁ -to C ₄ -alkylene and Q represents C ₁ -to C ₄ -alkyl- or C ₁ -to C ₄ -fluoroalkyl, the PQ group is bonded via P to the steroid backbone, R ¹¹ is halogen, R ¹³ is methyl or ethyl. The 7α-substituted 11β-haloestra-1,3,5 () according to claim 21, which is 11β-fluoro-3-hydroxy-7α-methylestra-1,3,5 (10) -trien-17-one. 10)-Trien. A nucleophilic substitution step using a halotalhydroxylation agent at position 11 in the 7α-substituted 11α-hydroxy steroid of Formula 4B; In this case using a step of reacting the produced 7α-substituted 11β-halogen steroid with an alkylating agent on a C ¹⁷ atom of the ring skeleton in a selective manner to form a 7α, 17α-substituted 11β-halogen steroid of Formula 10, 21. A 7α, 17α-substituted 11β-halogen steroid of formula 10 according to any one of claims 11 to 20 wherein UVWXYZ has a C ¹ -C ² -C ³ -C ⁴ = C ⁵ -C ¹⁰ ring structure. Way. A nucleophilic substitution step using a halotalhydroxylation agent at position 11 in the 7α-substituted 11α-hydroxy steroid of Formula 4B; In this case reacting the produced 7α-substituted 11β-halogen steroid with an alkylating agent on a C ¹⁷ atom of the ring skeleton in a selective manner to form a 7α, 17α-substituted 11β-halogen steroid of formula 10; Isomerizing the 7α, 17α-substituted 11β-halogen steroids of Formula 10 to form the corresponding isomers of Formula 12, wherein UVWXYZ is a C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ ring structure Using the steps The UVWXYZ produces a 7α, 17α-substituted 11β-halogen steroid of formula 12 according to any one of claims 11 to 20 in which the C ¹ -C ² -C ³ -C ⁴ -C ⁵ = C ¹⁰ ring structure. Way. A nucleophilic substitution step using a halotalhydroxylation agent at position 11 in the 7α-substituted 11α-hydroxy steroid of Formula 4B; In this case oxidizing the produced 7α-substituted 11β-halogen steroids to form 7α-substituted estradio-1,3,5 (10) -triene of formula 6 according to claim 17 or 18, Reacting the 7α-substituted estra-1,3,5 (10) -triene of formula 6 with an alkylating agent on a C ¹⁷ atom of the ring backbone in a selective manner to form a 7α, 17α-substituted 11β-halogen steroid of formula 8 Using steps, 21. A 7α, 17α-substituted 11β-halogen steroid of formula 8 according to any one of claims 11 to 20 wherein UVWXYZ has a C ¹ = C ² -C ³ = C ⁴ -C ⁵ = C ⁶ ring structure. Way. Use of a 7α, 17α-substituted 11β-halogen steroid of formulas 8, 10, 12 according to any one of claims 11 to 20 for the production of a pharmaceutical medicament. A pharmaceutical formulation containing at least one of the 7α, 17α-substituted 11β-halogen steroids of formulas 8, 10, and 12 according to any one of claims 11 to 20 and at least one pharmaceutically acceptable carrier.