EA008147B1 - Microbiological method for the production of 7 alpha-substituted 11 alpha-hydroxysteroids - Google Patents

Abstract

Disclosed is a microbiological method for the production of 7 α-substituted 11 α-hydroxysteroids general formal (4,B)in whichRis the grouping P-Q, wherebyP represents a C-C-alkylene, and Q represents a hydrogen, C-C-alkyl- or C-C-fluoroalkyl, and the grouping P-Q is bonded via P to the steroid skeleton,Rcan be in .alpha.- or .beta.-position and stands for H, CHor CF, andRis methyl or ethyl,from corresponding 7 α-substituted 17 β-hydroxysteroid by hydroxylating and oxydizing with use of a microorganism that is selected from the group that comprises Aspergillus sp., Beauveria sp., Glomerella sp., Gnomonia sp, Haplosporella sp. and Rhizopus sp.

Description

The present invention relates to microbiological methods for preparing 7a-substituted 11a-hydroxysteroids, to 7a, 17a-substituted Ιΐβ-halogen-steroids obtained from them, to methods for preparing the latter compounds, as well as to their use and to pharmaceutical preparations containing these compounds. In addition, the invention relates to other 7a-substituted 11β halogen steroids, specifically to 7a-substituted estra-1,3,5 (10) -trienes, derived from 7a-substituted 11 a-hydroxysteroids.

For the treatment of menopausal disorders in men (C11Tastegscht1e) and for the development of the male genital organs, as well as for the control of the reproductive ability (fertility) of men, androgens are used, primarily testosterone. In addition, these hormones also contain active ingredients with partially anabolic effects, which among other things contribute to muscle growth.

The climacteric period in men is characterized by a natural, caused by age-related changes in the body, a decrease in the production of androgens, to compensate for the lack of which, hormone replacement therapy (HRT) is used.

The introduction of LH (luteinizing hormone) and WG (releasing hormone) into the body to control male fertility, along with a decrease in spermatogenesis, also leads to excessive activation (rash) of LH and to a decrease in testosterone levels and extinction of libido, to increase which drugs containing testosterone (Ό.Ε. Sittshdk etc., Rgo51a1e-8rappd EGGesG og Ize Ro1ep1 Apygodep 7a Me1yu1-19-Yog1e81o81egope:. A Ro1epya1 Ayegpayue Yu Teijo ^ egope Gogh Apygodep Ver1asetep1 apy Ma1e Sop1gaser1yup, 1oita1 og S11shsa1 Epyosppo1odu apy Me ao1scht,! t. 83, No. 12, 1998, pp. 4212-4219).

Combination therapy using androgens and a gestagenic component can be used to control male fertility (see, for example, application XVO 01/60376 A, as well as the sources cited in this publication as a reference).

When treating with the use of testosterone, the occurrence of side effects, such as an increase in the prostate gland caused by an increase in the number of cells and stromal glands (benign prostatic hyperplasia, BPH), was found. In the metabolism of testosterone, mediated by 5a-reductase, dihydrotestosterone (DHT) is formed, which, among other things, can lead to BPH (Sittsdk et al., See above; application νθ 99/13883 A1). Therefore, in clinical practice, the suppression of 5a-reductase is used to treat BPH (the use of finasterides).

The rapid metabolism of androgenic steroid, which is testosterone, in the human body further leads not only to the formation of unwanted DHT, but also necessitates the oral administration of high doses to achieve the required effective level of testosterone. Therefore, there is a need to use alternative dosage forms, such as intramuscular injections or large patches.

To replace testosterone used in the above indications, 7amethyl-19-nortestosterone (MENT) was proposed, the biological effectiveness of which, firstly, is higher than testosterone, because it has a higher affinity for androgenic receptors, and which, secondly , forming a peculiar steric barrier due to the 7a-methyl group, prevents, as it is supposed, the process of metabolism under the action of 5a-reductase (Sittdk et al., see above; νθ 99/13883 A1; AO 99/13812 A1; I8 5342834).

In the metabolism of testosterone, in addition, some of this compound is transformed into estradiol as a result of aromatization of the ring A of the steroid system, especially in the brain, in the liver and in adipose tissue. Estradiol regarding the general effect of testosterone and its metabolites is to a decisive degree responsible for the properties and regulation caused by the specific nature of the sex and gonadotropins. Therefore, the effect of estradiol, as well as the effect of testosterone in the body of an adult man should be considered as a positive factor (Sittdk et al., See above).

However, it was found that the pharmacokinetics of testosterone is unsatisfactory. First of all, when administered orally, testosterone is rapidly eliminated from the body, as a result of which the effectiveness and duration of action of testosterone-based drugs also does not give satisfactory results. Therefore, a number of testosterone derivatives were synthesized. Such derivatives are described, in particular, in patent I8 5952319. First of all, it concerns 7a, 11β-dimethyl derivatives of 19-nortestosterone, such specifically as 7a, 11c-dimethyl-17v hydroxyestr-4-en-3-one, 7a, 11 b-dimethyl-17b-heptanoyloxyestr-4-en-3-one, 7a, 11 b-dimethyl-17b - [[(2cyclopentylethyl) carbonyl] oxy] estr-4-en-3-one, 7a, 11 b- dimethyl-17b- (phenylacetyloxy] estr-4-en-3-one and 7a, 11b-dimethyl-17β- | [(trans-4- [n-butyl] cyclohexyl) carbonyl] oxy] estr-4-ene -3-he

Named 7α, 11β-dimethyl derivatives possess, similarly to MeNT, the same advantages, including improved pharmacokinetics, in other words, their effectiveness and duration of action are elevated to a higher level compared to testosterone. However, a significant drawback of these derivatives is their requiring significant material and technical costs.

- 1 008147 synthesis.

One of the possibilities for the synthesis of steroids by microbiological means is described in EP 0900283 B1. This publication indicates that estr-4-en-3,17-dione and canrenone using a specific microorganism selected from the group comprising Akregd Shik shdpsapk, Ρΐιίζοριίδ aggIhih and strains Pc51c1oOa. can be transformed into the corresponding 11a-hydroxy-analogue. At the same time, in the introductory part of the description of the mentioned publication, reference is made to the work of ZYIAT and other authors in the US. Wurk Lasa 202, 1970, p. 172-179, which states that the microbially carried out reaction of 11a-hydroxylation of steroids refers to reactions that are not amenable to exact calculation.

Based on the foregoing, the present invention was based on the task of obtaining testosterone derivatives, which would be immune to recovery under the action of 5a-reductase, would have improved pharmacokinetics and above all would be characterized by simplicity and ease of their production. One of the extremely important aspects of the present invention consists accordingly in the development in order to simplify the production of part-products in such a way that would allow to get these part-products without any problems.

The present invention is based on microbiological methods for producing 7a-substituted steroids according to claims 1, 3 and 6 of the claims, using 7a, 17a-substituted Πβ-halogen steroids according to claim 11, methods of obtaining 7a, 17a due to the use of these 7a, 17a-substituted Πβ-halo-steroids according to claim 26, thanks to pharmaceutical preparations containing these 7a, 17a-substituted Πβ-halogen steroids, according to clearly to claim 27 of the claims and with help of 7a-substituted-galogenestra Πβ-1,3,5 (10) trienes according to claim 21 of the claims. Preferred embodiments of the invention are presented in the dependent claims.

Explanations

The following explanations apply to all sections of the description and to the claims, as well as to the accompanying scheme I.

All groupings, residues and other structural units may, respectively, independently of each other vary in the specified ranges of values.

All alkyl, alkylene, alkenyl, alkenylene, alkynyl, and alkynylene groups can have either a straight or branched chain. For example, the propenyl group can be represented as the following chemical structures: -CH = C-CH ₃ -CH ₂ -C = CH ₂ and -C (CH ₃ ) = CH ₂ . Thus, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, i-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 1- methyl n-pentyl, 2-methyl-n-pentyl, 3-methyl-n-pentyl, 4-methyl-n-pentyl, 1-ethyl-n-butyl, 2-ethyl-n-butyl, etc.

An alicyclic alkyl is either cycloalkyl or a cycloalkyl substituted by one alkyl group or several alkyl groups, which is attached directly through a cycloalkyl ring or through one of the alkyl groups.

Similarly, an alicyclic alkenyl is either cycloalkenyl, or substituted by one or more alkenyl groups or one or more alkenyl and alkyl groups, or one or several alkyl groups cycloalkenyl or cycloalkyl, which is attached directly through a cycloalkenyl ring or through one of the alkenyl or through one of the alkyl groups, while in the alicyclic alkenyl contains at least one double bond.

Aryl can be phenyl, as well as 1-naphthyl or 2-naphthyl. The term aryl also includes, in principle, heteroaryl, especially 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, as well as 3- and 4-pyridazinyl.

Halogen is fluorine, chlorine, bromine or iodine.

Pharmaceutically acceptable additive salts are the salts of the corresponding compounds with inorganic and organic acids, such as, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, citric acid, oxalic acid, tartaric acid and methanesulfonic acid. Esters can be formed primarily with succinic acid.

Numbers printed in superscript with the appropriate character B, for example B ¹³ , designate its position in the steroid skeleton, while the C-atoms in this skeleton are numbered according to the IUPAC nomenclature. Numbers printed as superscript with the corresponding character C, for example C ^ten , denote the position of the corresponding carbon atom in the steroid skeleton.

- 2 008147

Description of the invention

Microbiological methods of the new type are designed to obtain 7a-substituted 11α-hydroxysteroids of general formula 4, B

wherein

AT ⁷ is a grouping of pp, where

P stands for C _one -WITH _four alkylene, and C) denotes hydrogen, C _one -WITH _four alkyl or C _one -WITH _four fluoroalkyl (alkyl partially or fully fluorinated), and this group P ^ through P is linked to the steroid skeleton,

AT ^ten represents H, CH ₃ or CP ₃ and

AT ¹³ represents methyl or ethyl.

In one of the first variants of the method of obtaining these compounds, the corresponding 7α-substituted steroid of the general formula 3, A

in which ⁷ , AT ^ten and B ¹³ have the same meanings as those indicated for the compound of general formula 4, B, using an appropriate microorganism selected from the group comprising AzregdSiz sp., Veaiuepa sp., Oteotera sp., Opota sp., Narozroge 11a sp. and 1BH / oryz sp., at the appropriate stage of the process, are hydroxylated and oxidized. Particularly preferred AzregdShiz yszyep (ATSS 1020), AzregdShiz taydpiz (ΙΜΙ 16061), AztsdSchiz psheg (ATSS 9142), Veyuyepa Oztsv (otstp 7159), O1terer 11a stdi1a! ), Gadget stryYa (CB8 15226), Narozroge11a Nezregebyuya (CB8 20837) and YOU / oriz zlotorot! (ATSS 15441).

This microbiological method can also be carried out by a two-stage mechanism, sequentially carrying out the hydroxylation reaction and the oxidation reaction. The course of the reaction can be controlled by its duration. For example, by stopping the reaction after a certain period of time, it is possible to isolate hydroxylated, but not yet oxidized products. Both of these stages can therefore be carried out separately or by a mixed enzymatic reaction.

To this end, the compound of general formula 3, A is in the first stage of the microbiological method using one of the microorganisms selected from the group consisting of Azregdgir sp., Veaiuepa sp., O1bene11a sp., O1otege11a sp., Opoto sp., Meragon17sht sp. sp, YOU / ory sp. and Hygienic sp., are hydroxylated at position 11 with the formation of a 7a-substituted steroid with a hydroxy group at position 11. This compound corresponds to the general formula C

- 3 008147

in which K ⁷ To ^ten and K ¹³ have the same meanings as indicated above for compounds of general formula 4, B. Above all use of the following microorganisms: Aspergillus taydpiz, te11eiz Aspergillus, Aspergillus P1§eg, Aspergillus osygaseiz, Veaiuepa azz1apa, O1ege11a Gitskigots O1ege11a / eae, O1otege11a stdi1a1a, O1otege11a Gizago1bez, Opotota stdi1a1a, Me1agg1n / 1it atzorNae, Yschgozroga zryaepsa, Rbl / OSU opus / ae, KS / oryz z1O1ot Geg and UgysShsht bayayae. Most preferably, for the hydroxylation of Aspergillus taydpiz (ΙΜΙ 16061), Aspergillus te11eiz (CD8), Aspergillus tdeg (ATCC 11394), Aspergillus osygaseiz (YKK 405 CD8 13252, ATCC 46504), Veaiuepa azz1apa (ATCC 7159, ΙΡΟ 5838, ATCC 13144, ΙΡΟ 4848, SV8 11025, SV8 12736), Oliver 11a Kirkigo1 (ATSS 14842), Oliver 11a / ee (SV8 4474), Oiotega 11a shtsi1a! , ΙΡΟ 6470, CB8 98069, ΙΡΟ 7478, ΙΡΟ 5257, ATCC 64682, ATCC 15470 1 О от СС 1,,,,,,,,,,,,,,,,,,,,,,,,, М А А А еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп СВ СВ СВ СВ СВ СВ СВ СВ СВ еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп еп oriz ogu / ae (AT SS 4858, ATSS 34102, ATSS 34102), KY / Orizal Opportunity (ATSS 6227b, ATSS 15441) and WeissTaKeKe (ATSS 11405).

Upon completion of the hydroxylation, an intermediate product of the formula C in the second stage of the microbiological method using another microorganism selected from the group including Wasciz sp., Musobaepit sp., Bosagba sp. and Rzeibotopaz sp., are oxidized to form as a result of 7a-substituted 11-a-hydroxysteroids of general formula 4, B. For this purpose use primarily the following microorganisms: Bacillus 1asytogiz, Bacillus zryaepsiz, MusoaOepit peoaigit, Musoas1elit ztedtayz, Yosagb1a sogaShpa, Yosagb1a d1oegi1a, Yosagb1a tSh1ta, Yosagb1a gezygsShz, Yosagb1a gigoregypOa, Yosagb1a za1totso1og and Rzeibotopaz 1ez1oz1egop1 most preferably used Bacillus 1asytogiz ( ATSS 245 ttt (ATSS 19150), Bosagbondurus (SSCH 10027), Boscaglinauscoregio (ATCC 14352), Boscabla1 (Totcovicum (ATCC 19149), and Rezebothapazus Radiophilus (ATCC 11996).

According to another variant of the method, compounds of the general formula 4, B can be obtained by

in which K ⁷ To ^ten and K ¹³ have the same meanings as indicated for compounds of general formula 4, B. This reaction is carried out with the use of an appropriate microorganism selected from the group including AzregdShiz sp., Veaiuyeroma zyr, Sigui1apa zr., Olyberzha spy / oryz sp. and Supersialagit sp., while the steroid carcass is hydroxylated at the 11α position, and the result is a 7a-substituted 11αhydroxysteroid of general formula 4, B. Preferred for this purpose the following microorganisms: Aspergillus aShaseiz, Aspergillus amatol, Aspergillus PzsIep, Aspergillus taydpiz, Aspergillus te11eiz, Aspergillus tbia1apz, Aspergillus tdeg, Aspergillus osygaseiz, Aspergillus uapeso1og, Veaiuepa azz1apa, Sigui1apa 1ipa1a, O1ege11a / eae, O1otege11a stdi1a1a, O1otege11a 1izago1bez, Optotech aaaaaaaaaaa SS 1020), Aspergillus taydpiz (Μ 16061), Aspergillus te11eiz (CD8), Aspergillus tbia1apz (ATCC 11267), Aspergillus tdeg (ATCC 9142, ATCC 11394), Aspergillus osygaseiz

- 4 008147 (ΝΚΚΕ 405, ATCC 13252, ATCC 46504), AzregdDIz-4994 (ATSS 10067), Veaiuepa AZzza (GROZ 5838, ATSS 13144, 1RO 4848, SV8 11025, CB8 12736, and 14444), 1444, 474, APTS 13144, 1RO 4848, CB82525 / eae (CD8 4474) O1otege11a eshdiYa (ATCC 10534, CD8 23849, CD8 23749, ATCC 16646, 1RO 6459, 1RO 6425, 1RO, 6470, ATCC 15093, ATCC 10529, 1RO 5257, ATCC 56596, ATCC 64682), O1otege11a GizagoLez (ATSS 9552), Opotosh stgl1a1a (CB8 15226), Narozogroze 11zregeLea (SV8 20837), Ne11soz11it rtGogtae (ATSS 8992), №grozrah zrIaepsa (ATSS 12 ha, ian jar of 4827).

The most suitable are the methods by which 7a-substituted 11α-hydroxysteroids of the general formula 4, B are obtained, where K is independent of ⁷ represents CH3 and / or K ^ten is H and / or K ¹³ represents CH3.

The method is carried out in the usual manner. Usually, a sterilized nutrient solution is first prepared for the strain, and then the culture solution containing it is introduced into the nutrient solution to culture the strain. Next, the preliminary culture thus obtained is transferred to a fermenter, into which the corresponding nutrient solution is also loaded. Preferably, after the initial stage of growing the culture of the strain, the starting material is then added to the fermenter, in this case either a compound of general formula 3, A, or a compound of general formula Ό, thus initiating the reaction proposed in the invention. Upon completion of the reaction, the mixture of substances to highlight the desired 7a-substituted 11 α-hydroxysteroid is purified by conventional technology.

From the compounds of general formula 4 obtained in this way, B, it is possible to synthesize other compounds proposed in the invention using the methods also proposed in the invention. First of all, 7a, 17a-substituted 11c-halogen-steroids of the general formula 8,10,12

wherein

IU-SH-Χ-Υ-Ζ is one of the cyclic structures C ^one -WITH ² -WITH ³ -WITH ^four = C ^five -WITH ^ten , WITH ^one -WITH ² -WITH ³ -WITH ^four WITH ^five = C ^ten or with ^one -WITH ² -WITH ³ -WITH ^four -WITH ^five -WITH ^ten , while in this case, the oxo group (= O) is associated with \ Ш (С ³ ) or is a cyclic structure C ^one = C ² -WITH ³ = C ^four -WITH ^five = C ⁶ , in this case, the remainder is OK ³ associated with W (= C ³ ),

TO ³ represents H, C1-C _four alkyl, C1-C _four alkanoyl or simple cyclic C ₃ -WITH ₇ ether with OATOM residue OK ³ ,

TO ⁷ is a grouping of pp, where

P stands for C1-C _four alkylene, and P denotes hydrogen, C1-C _four alkyl or C1-C _four fluoroalkyl (alkyl partially or fully fluorinated), and this group P-P through P is linked to the steroid skeleton,

TO ^ten may be in the α- or β-position and represents H, CH ₃ or CP ₃ however, it is present only under the condition that Χ-Υ-Ζ does not mean C ^four -WITH ^five = C ^ten ,

TO ^eleven is a halogen,

TO ¹³ represents methyl or ethyl,

TO ¹⁷ represents H, C _one -WITH ₁₈ alkyl, alicyclic C _one -WITH ₁₈ alkyl, C _one -WITH ₁₈ alkenyl, alicyclic C _one -WITH ₁₈ alkenyl, C _one -WITH ₁₈ quinil,

WITH _one -WITH ₁₈ alkylaryl, C _one -WITH _eight alkenylamine or group P-P, where this group P-P has the above value,

TO ¹⁷ is H, C1-C18 alkyl, alicyclic C1-C18 alkyl, C1-C18 alkenyl, alicyclic C1-C18 alkenyl, C1-C18 alkynyl or C1-C18 alkylaryl, while K ¹⁷ may also be via the keto group associated with the 17v-hydroxy group and, moreover, K ¹⁷ may additionally be substituted by one or several groups #K. ¹⁸ TO ^nineteen or one or several groups 8OHK ²⁰ where x denotes 0, 1 or 2, and K ¹⁸ , 19 20 17

K and K, each independently of one another, can have the same meaning as K, as well as their pharmaceutically acceptable addition salts, esters and amides are active substances that have a number of important advantages. These are obtained as a result of carrying out other stages of the method of 7a-substituted 11 α-hydroxysteroid of general formula 4, B compounds are valuable active substances with a pronounced androgenic effect, without showing the above-mentioned side effects when using them. These compounds are suitable for

- 5 008147 appropriate medicines, and above all effective contraceptives and active substances for hormone replacement therapy (HRT).

In the case of additional substitution K ¹⁷ group ΝΚ ¹⁸ Κ ^nineteen it can be a methylamino, dimethylamino, ethylamino, diethylamino, cyclohexylamino, dicyclohexylamino, phenylamino, diphenylamino, benzylamino or dibenzylamino group.

The 7a, 17a-substituted Ιΐβ-halogen steroids of the general formula 8,10,12 that are especially suitable for use for these purposes include compounds in which C-Y - ^ - Χ-Υ-Ζ is a cyclic structure C ^one -WITH ² -WITH ³ -WITH ^four = C ^five -WITH ^ten , WITH ^one -WITH ² -WITH ³ -WITH ^four -WITH ^five = C ^ten or with ^one = C ² -WITH ³ = C ^four -WITH ^five = C ^ten .

In the first case (C-Y - ^ - Χ-Υ-Ζ means C ^one -WITH ² -WITH ³ -WITH ^four = C ^five -WITH ^ten ) we are talking about steroids of General formula 10

In the second case (I-Y - ^ - Χ-Υ-Ζ means C ^one -WITH ² -WITH ³ -WITH ^four -WITH ^five = C ^ten ) we are talking about steroids of General formula 12

The compounds of general formulas 10 and 12 are compounds with androgen action.

In the third case (C-Y - ^ - Χ-Υ-Ζ means C ^one = C ² -WITH ³ = C ^four -WITH ^five = C ^ten ) we are talking about steroids of General formula 8

These compounds are estrogens (compounds with affinity for estrogen receptors).

In all three cases, residues K ³ To ⁷ To ^ten To ^eleven To ¹³ To ¹⁷ and K ¹⁷ have the same meanings as the corresponding residues in general formula 8, 10, 12.

Preferably, independently of each other, K ^one is H and / or K ⁷ represents CH3, and / or K ^eleven is fluorine, and / or K ¹³ represents CH3, and / or K ¹⁷ represents H, CH ₃ , WITH _one -WITH ₁₈ quinil, especially ethinyl, CH ₂ С№ or СР ₃ and / or K ¹⁷ represents N.

Particularly suitable for use in the invention 7a, 17a-substituted 11 β halogen steroids of the general formula 8,10,12 include the following compounds:

17a-ethynyl-11b-fluoro-17b-hydroxy-7a-methylestr-4-en-3-one (formula 10), 17a-ethynyl-11b-fluoro-17b-hydroxy-7a-methylestr-5 (10) -en -3-one (formula 12), 17a-ethynyl-11c-fluoro-7a-methylestra-1,3,5 (10) -triene-3,17b-diol (formula 8).

These compounds can be synthesized in the following way:

For 7a, 17a-substituted Πβ-halogen-steroids of general formula 10, where C-Y is ^ - ^-Υ-Ζ

- 6 008147 is a cyclic structure C ^one -WITH ² -WITH ₃ -WITH ^four = C ^five -WITH ^ten , as starting materials, the 7a-substituted 11αhydroxysteroids of general formula 4, B, obtained using the microbiological method of the invention, are used.

In the first stage of the synthesis, the 7a-substituted 11 α-hydroxy steroids thus obtained are converted by a nucleophilic substitution with a halodehydroxylation reagent into the corresponding

As can be seen galodegidroksiliruyuschih reagents commonly used for all these purposes the compounds, such as hydrofluoric, hydrochloric, hydrobromic or hydroiodic acid, thionyl chloride or thionyl bromide, phosphorus pentachloride, phosphorus oxychloride, Νhlorsuktsinimid, triphenylphosphine / tetrachloromethane, HE / pyridine or trifluoride diethylaminosulfur or preferably nonaflifluoride / 1,5-diazabicyclo [5.4.0] undecen.

From the compound of formula 5 can then by selective alkylation at the atom C ¹⁷ framework to obtain the compound of formula 10 (see scheme I). Conventional alkylating reagents can be used for selective alkylation, for example, Grignard compounds and organometallic compounds, especially alkyl lithium compounds. For example, to obtain the corresponding 17a-ethynyl17p-hydroxy-estr-4-en-3-one from estr-4-ene-3,17-dione, ethynyl magnesium bromide can be used as an alkylating reagent.

For 7a, 17a-substituted 11 β-halogen-steroids, in which ϋ-ν - ^ - Χ-Υ-Ζ is a cyclic structure C ^one -WITH ² -WITH ³ -WITH ^four -WITH ^five = C ^ten and which correspond to the general formula 12, compounds of the general formula 10 are used and isomerized, as a result of which the double bond Δ ^four isomerized into a double bond Δ ^{5 (10)} . To protect the 3-keto groups, they first form a cyclic ether at position 3. Then the double bond Δ ^four isomerized into a double bond Δ ^{5 (10)} with the formation of 7α, 17α-substituted 11 β-halogen-steroid of general formula 12, after which the protective group is again cleaved off.

Upon receipt of the other 7a, 17a-substituted 11 β-halogen steroids of the general formula 8, where ϋ-ν - ^ - ΧΥ-Ζ is the cyclic structure C ³ = C ² -WITH ³ = C ^four -WITH ^five = C ^ten , work as follows:

First, as already described above, from the resulting microbial hydroxylation and oxidation of 7a-substituted 11α-hydroxysteroid of general formula 4, B by halodehydroxylation by the nucleophilic substitution reaction form the corresponding 11β-halogen steroid of general formula 5.

Then, from this 11 β-halogen-steroid by oxidation, for example, with the salt of copper (11), form 7 α-

in which ³ , AT ⁷ , AT ^eleven and B ¹³ have the above values. If in ³ represents H, then these compounds can be synthesized directly.

If B ³ matters different from H, then after the formation of the 1,35 (10) triene ring by oxidation, it is necessary to form the corresponding ethers and esters by the usual method.

7α-substituted 11β-halogenestra-1,3,5 (10) -trienes of general formula 6, as well as their pharmaceutically acceptable additive salts, esters and amides, are new and are therefore included in the scope of the present invention as intermediate products used in the synthesis is also proposed in the invention 7α, 17α-substituted 11 β-halogen steroids of General formula 8.

One of the particularly preferred 7α-substituted 11β-halogenestra-1,3,5 (10) -triene total

- 7 008147 of formula 6 is 11c-fluoro-3-hydroxy-7a-methylestra-1,3,5 (10) -trien-17-one.

From 7a-substituted 11c-halogenestra-1,3,5 (10) -triene of the general formula 6, similar to the synthesis of the compound of the general formula 10 described above by selective alkylation at the C atom ¹⁷ the skeleton can be formed as proposed in the invention 7a, 17a-substituted Πβ-halogen steroid of general formula 8.

In addition, from the resulting microbial hydroxylation and oxidation from 7a-substituted steroids of general formula 3, A or Ό of substances of general formula 4, B, 7a-substituted Πβ-halogen-steroids of general formula 9 with the androgenic effect can also be obtained:

in which K ⁷ To ^eleven and K ¹³ have the above values. One of the particularly preferred compounds is 11c-fluoro-17c-hydroxy-7a-methylestr-4-en-3-one. The compounds of general formula 9, as well as their pharmaceutically acceptable addition salts, esters and amides, also have an androgenic effect.

To obtain compounds of general formula 9, estr-4-ene-3,17-dione of formula 5 is reduced to 17c-hydroxyestr-4-en-3-one of formula 9, for example, using the corresponding borohydride.

The compounds of general formula 9 can also be converted into the corresponding 7a-substituted 11c-halogenestra-5 (10) -enes of formula

in which K ⁷ To ^ten To ^eleven and K ¹³ have the same meanings as those indicated in the general formula 8,10,12. To this end, compounds of general formula 9 are isomerized by displacing the double bond Δ ^four in double bond Δ ^{5 (10)} . To protect the 3-keto groups, they first form a cyclic ether at position 3. Then the double bond Δ ^four isomerized into a double bond Δ ^{5 (10)} with the formation of the above 7 α-substituted 11 β-halogen-steroid, after which the protective group is cleaved again.

Another possibility to obtain the corresponding 7a-substituted 11c-halogenoestra-5 (10) -enes of the formula

in which K ⁷ To ^ten To ^eleven and K ¹³ have the same meanings as those indicated in the general formula 8,10,12, is that they are formed from compounds of the general formula 5 due to the isomerization of the double bond Δ ^four in double bond Δ ^{5 (10)} . To this end, compounds of general formula 5 are isomerized by displacing the double bond Δ ^four in double bond Δ ^{5 (10)} . To protect the 3-keto groups, they first form a cyclic ether at position 3. Then the double bond Δ ^four isomerized into a double bond Δ ^{5 (10)} with the formation of the above 7a-substituted 11β-halogen-steroid, after which the protective group is cleaved again.

All of these compounds can also be esterified further to form esters or ethers, but only if there are corresponding hydroxy groups in position 3 or 17β. So, for example, a compound of formula 9 can be converted to the corresponding Ηβ-ether or complex

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17β-3φπρ. One of the preferred compounds of this group is 11β-φτορ-17β- (4sulfamoylbenzoxy) -7a-methylestr-4-en-3-one. As substituents on the oxygen atom of the hydroxy group at C ¹⁷ basically the same residues as indicated for B are suitable. ¹⁷ .

First of all, 7a, 17a-substituted 11β-halogen-steroids of the general formula 8,10,12 are suitable for the preparation of corresponding medicinal products. With this in mind, the present invention also relates to the use of the said compounds of general formula 8,10,12 for the preparation of corresponding medicinal products, as well as pharmaceutical preparations containing at least one of the indicated compounds of general formula 8,10,12 and at least one pharmaceutically acceptable carrier.

The 7a, 17a-substituted 11 β-halogen-steroids of the general formula 10,12 according to the invention are compounds with a pronounced androgenic effect, without showing the above-mentioned side effects, such as, for example, prostate gland stimulation (above all, benign hyperplasia prostate). The advantages of the compounds include their simple synthesis. It was established that the compounds of the general formula 10 or 12 proposed in the invention can be used not only for men’s HRT, they are also suitable without the additional introduction into the body of any other active substances as effective male contraceptives with appropriately selected dosage in order to reduce the level of in the blood of LH, produced in the body of testosterone and FSH (follicle-stimulating hormone). This is due to the ability of the 11β halogen steroids according to the invention to inhibit the excessive activation (rash) of LH and FSH. LH stimulates Leydig cells in such a way that they secrete testosterone. If the content of LH in the blood is kept low, then the release of testosterone in the body also decreases. Testosterone is used for spermatogenesis, whereas FSH stimulates germ cells. Therefore, for effective spermatogenesis, a sufficiently high level of FSH and LH in the blood is required, while a sufficiently high level of LH in the blood causes excessive activation of testosterone, which is necessary for spermatogenesis.

Since the use of only 7a, 17a-substituted 11β halogen steroids for sterilization without the addition of any additional active ingredients can already lead to effective male contraception, this will not only significantly simplify the administration of the appropriate medicinal product intended for these purposes, but also significantly reduce costs associated with its use.

To control male fertility, the 7a, 17a-substituted 11 β halo-steroids according to the invention can also be used in combination with a gestagen.

In addition, the 7a, 17a-substituted 11β-halogen steroids according to the invention effectively inhibit 5a-reductase and the steroid 11-hydroxylase [SUR11B (P450c11). Ζ1ιαη§. Mb MSHEG, 1goo1 oh! S11P1sa1 Eibosgshodod apb MeGaOukt, vol. 81, 1996, p. 3254-3256], due to which it is possible, for example, to selectively avoid a stimulating effect on the prostate gland, and the compounds exhibit improved pharmacokinetics. Inhibition of 11-hydroxylase leads to a decrease in the degree of deactivation of compounds with androgenic effect and to a decrease in their release from the human body. This increases the efficiency and duration of action of these compounds in comparison with known compounds, especially when administered orally.

The above properties allow the compounds proposed in the invention to be used primarily to control the reproductive ability of men, as well as for androgen replacement therapy with a reduced tendency to restore 5a-reductase while maintaining aromatization with the formation of estrogenic steroids and a positive effect on serum lipids and the central nervous system.

The androgenic effect and the absence, as it was previously established, of the above-mentioned side effects was investigated in experiments on the seminal vesicles using the compounds of general formulas 10 and 12 proposed in the invention. The effectiveness of the compounds of general formula 8 proposed in the invention was tested in an experiment on the estrogenic effect that these compounds have on the growth of the uterus.

The 7a, 17a-substituted 11 β-halogen-steroids of the general formula 10 or 12 proposed in the invention, as well as the pharmaceutical preparations according to the invention containing these compounds, are suitable for exclusively successful treatment of non-infertile men, as well as, in principle, male mammalian animals. . The use of male contraception for men only leads to a temporary loss of reproductive ability by men. Upon completion of taking the active ingredients of the invention, respectively, of pharmaceutical preparations, the initial state is restored, i.e. reproductive ability returns to the man and spermatogenesis proceeds in the same volume as before. To achieve temporary sterility and maintain it at a constant level for the required period of time, the active ingredients, respectively, the drug must be administered into the body continuously, without interruption, and depending on the form of their application

- 9 008147 should be prescribed daily, and in other cases, provide for periodic repetition after certain short or long periods of time. After a single or multiple administration of the active ingredient or drug, the restoration of the initial non-sterile state of the man does not necessarily happen immediately, it can be restored gradually, which will require a certain period of time depending on various factors, such as dosage, features of the human constitution and simultaneous administration other drugs.

If the purpose of use is contraception, then the dosage of 7a, 17a-substituted 11 β halosteroids should be chosen so high that the content of LH and FSH in the blood is respectively a maximum of 2.5 IU / ml, first of all a maximum of 1.0 IU / ml, was a maximum of 10 nmol / l, first of all a maximum of 3 nmol / l.

If the proposed 7a, 17a-substituted 11 β-halogen-steroids in the invention are to be used for HRT without achieving contraception, then the dosage should be chosen lower. In this case, they strive to achieve a level of active substances that provides the content of LH and FSH in the blood, respectively, above 2.5 IU / ml, and the testosterone content is above 10 nmol / l.

The dosage of the proposed in the invention 7a, 17a-substituted 11 β-halogen steroids of general formula 10 or 12, required for the regulation of blood levels of LH, FSH and testosterone, depends on many factors and should therefore be determined on the basis of the specific application. First of all, the dosage depends, obviously, on the type of therapy. If the compounds are supposed to be used for male contraception, they must be administered in significantly higher doses than when used for HRT. The dosage is further determined by the type of 7a, 17a-substituted Πβ-halogen steroid and its bioavailability. The specificity of the application is also extremely important for the choice of the amount of compound to be entered. In addition, the dosage depends on the constitution of a person and on other factors, such as, for example, the need to take into account the possible parallel intake of other medicines.

The compounds may be administered for oral and parenteral administration, for example, intraperitoneally, intravenously, intramuscularly or transdermally. Connections can also be implanted into the tissue. The amount of compound administered to an organism can vary widely, given the effectiveness of that amount. It can vary widely also depending on the condition to be treated and the method of administration. The daily dose for a person is from 0.1 to 100 mg. The preferred daily dose for a human is from 0.1 to 10 mg. The duration of use of the corresponding compound is determined by the ultimate goal.

Capsules, pills, tablets, dragees, creams, ointments, lotions, fluids, such as syrups, gels, injectable fluids, for example, for intraperitoneal, intravenous, intramuscular or percutaneous injection, etc., can be intended for practical use. the forms release the compounds of the invention, depending on their type, gradually or release the entire amount over a short period of time.

For oral administration, capsules, pills, tablets, dragees, and liquids or some other oral dosage forms are used as pharmaceutical preparations. In these cases, such drugs can be made in such a way as to ensure the release of active substances by them either after a short period of time, or to provide a depot effect, which will allow the active substance to enter the body gradually, with slowing down (prolonged action). Along with 7a, 17a-substituted Πβ-halogen steroid, unit doses may contain one or more pharmaceutically acceptable carriers, for example, substances for regulating the rheological properties of a drug, surfactants, hydrotropic solubilizers, microcapsules, microparticles, granules, diluents, binders, such as starch, sugar, sorbitol and gelatin, as well as fillers such as silicic acid and tal, lubricants, dyes, flavors and other substances.

The 7a, 17a-substituted Πβ-halogen steroids according to the invention may also be presented as a solution intended for oral administration and containing, along with the active Πβ-halogen steroid, as other components, a pharmaceutically acceptable oil and / or a pharmaceutically acceptable lipophilic, surfactant, and / or a pharmaceutically acceptable hydrophilic surfactant and / or a pharmaceutically acceptable water-miscible solvent. In this regard, reference may also be made to application XV O 97/21440.

To achieve the best bioavailability of the steroid, the compounds can be used to prepare cyclodextrin clathrates. For this purpose, the compounds are reacted with α-, β- or γ-cyclodextrin or with their derivatives.

If it is envisaged to use creams, ointments, lotions and other liquids intended for external use, these drugs should be formulated in such a way as to ensure that the compounds of the invention are able to enter the body in sufficient quantities. The composition of such dosage forms include certain excipients, on

- 10 008147 example of a substance for regulating rheological properties, surfactants, preservatives, hydrotropic solubilizers, diluents, substances to increase the ability of the steroids according to the invention to penetrate the skin, dyes, fragrances and skin protection agents such as air conditioners and moisture regulators. The composition of the drug along with the steroids according to the invention may include other active substances.

For parenteral administration, the active substances can be dissolved or suspended in an appropriate physiologically compatible diluent. In many cases, oils with or without the addition of a hydrotropic solubilizer, surfactant, suspending or emulsifying agent are used as diluents. Examples of oils used for these purposes include olive oil, peanut oil, cottonseed oil, soybean oil, castor oil, and sesame oil. For the preparation of an injectable preparation, any liquid carrier can be used, in which the compounds according to the invention are in dissolved or emulsified form. Such liquids often also contain viscosity regulating agents, surfactants, preservatives, hydrotropic solubilizers, diluents, and other additives that make the solution isotonic. Together with 7α, 17α-substituted Ιΐβ-halogen-steroids, other active substances can also be injected into the body.

The 11 β-halogen steroids according to the invention can be used as a depot injection or an implantable preparation, for example subcutaneously, which can be shaped in such a way as to ensure a gradual, slow release of the active substance. For this purpose, known technologies can be used, for example, depot forms that dissolve or are equipped with a membrane. Implants may contain as inert materials, for example, biodegradable polymers or synthetic silicones, for example silicone rubber. With percutaneous administration, the 11 β-halogen steroids according to the invention can be embedded, for example, in a patch.

Below the invention is explained in more detail by examples.

A. Microbiological synthesis

11a-hydroxy-7a-methylestr-4-ene-3,17-dione (compound 4, B)

Example 1

In a 2-liter Erlenmeyer flask after loading 1000 ml of a nutrient solution sterilized for 30 min at 121 ° C in an autoclave containing 3 wt.% Glucose, 1 wt.% Liquid obtained after soaking the corn kernels before swelling, 0.2 wt.% ΝαΝΟ ₃ , 0.1 wt.% KN ₂ Ro _four , 0.2 wt.% To ₂ NRA _four , 0.05 wt.% KC1, 0.05 wt.% Md8O _four -7H ₂ O and 0,002 wt.% Ge8O _four -7H ₂ About (pH 6.0), the culture of the strain Opoch etdi1a1a grown on stubble agar (SB8 15226) was inoculated and shaken on a rotary shaker at 165 rpm for 72 hours at 28 ° C. Then this preliminary culture was added to a 20-liter fermenter containing 19 liters of sterile medium of the same final composition as described above for the preliminary culture. In addition, prior to sterilization, another 1.0 ml of silicone oil and 1.0 ml of more pure (keto alcohol ethoxylate) were added to suppress pricing. At the end of the initial growth phase, which lasted for 12 hours at an elevated pressure of 0.7 bar, a temperature of 28 ° C, aeration at a flow rate of 20 l / min and a stirring speed of 250 rpm, a solution of 4.0 g of 17c-hydroxy was added. 7a-methylestr-4-en-3-one in 40 ml DMF. Then continued mixing and aeration. After 135 hours, the culture broth was collected and extracted for 10 hours with 10 liters of methyl isobutyl ketone and within 5 hours of 5 liters of methyl isobutyl ketone. The combined organic phases were concentrated to dryness. Silicone oil was removed by washing with hexane. After chromatography on silica gel using a hexane / ethyl acetate solvent gradient, 1.64 g (39%) of 11a-hydroxy-7-methyl-estr-4-ene-3,17-dione was isolated.

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Example 2

In a 2-liter Erlenmeyer flask after loading 1000 ml of a nutrient solution sterilized for 30 min at 121 ° C in an autoclave containing 3 wt.% Glucose, 1 wt.% Liquid obtained after soaking the corn kernels before swelling, 0.2 wt.% ΝαΝΟ ₃ , 0.1 wt.% KN ₂ Ro _four , 0.2 wt.% To ₂ NRA _four , 0.05 wt.% KC1, 0.05 wt.% Mg8O _four -7H ₂ O and 0,002 wt.% Re8O _four -7H ₂ About (pH 6.0), cultures of strain O1oshegela 11a S1PigiAla (1PO 6425) grown on beveled agar were inoculated and shaken on a rotary shaker at 165 rpm for 72 hours at 28 ° C. Then this preliminary culture was added to a 20-liter fermenter containing 19 liters of sterile medium of the same final composition as described above for the preliminary culture. In addition, before sterilization, another 1.0 ml of silicone oil and 1.0 ml of napregregop (ketospirit ethoxylate) were added to suppress pricing. At the end of the initial growth phase, which lasted for 12 hours at an elevated pressure of 0.7 bar, a temperature of 28 ° C, aeration at a flow rate of 20 l / min and a stirring speed of 350 rpm, a solution of 2.0 g of 17c-hydroxy was added. 7a-methylestr-4-en-3-one in 30 ml of DMF. Then continued mixing and aeration. After 19 hours, the culture broth was collected and extracted for 20 hours with 20 liters of methyl isobutyl ketone and for 23 hours with 20 liters of methyl isobutyl ketone. The combined organic phases were concentrated to dryness. The residue was almost completely dissolved in methanol. Silicone oil was filtered. Then it was concentrated and after chromatography on silica gel using a solvent gradient of dichloromethane / acetone, 1.55 g (73%) of 11a, 17c-dihydroxy-7a-methylestr-4-en-3-one was isolated. After recrystallization from acetone / diisopropyl ether, 827 mg (39%) of white crystals were isolated with a melting point of 163 ° C and [α] _ο = -16 ° (СНС1 ₃ c = 0.501).

In a 2-liter Erlenmeyer flask after loading 500 ml of a nutrient solution sterilized for 30 minutes at 121 ° C in an autoclave containing 0.5% by weight of glucose, 0.5% by weight of yeast extract, 0.1% by weight, into it. peptone and 0.2 wt.% of the liquid obtained after soaking the corn kernels before swelling (pH 7.5), four obtained by cryogenic ball culture of the strain Vaschiz sryepsiz (ATCC 7055) and shaken on a rotary for 24 hours at 28 ° C shaker at 165 rpm This pre-culture was then introduced into four 2-liter Erlenmeyer flasks containing 500 ml of a sterile medium of the same composition as described above for the pre-culture, while 10% of the culture broth was inoculated into each flask. At the end of the initial growth phase, which lasted for 4 hours at a temperature of 28 ° C using a rotary shaker at 165 rpm, 50 mg of 11a, 17c-dihydroxy-7a-methylestr-4-en-3ona was added to each of the four flasks in 2.5 ml DMF. The shaking was continued for another 48 hours. The combined culture broths were extracted twice with 2 L of methyl isobutyl ketone. The combined organic phases were dried over sodium sulfate and concentrated to dryness. 630 mg of an oily crystalline residue were obtained. After recrystallization from acetone / diisopropyl ether, 103 mg (49.2%) of yellowish crystals were isolated with a melting point of 189 ° C and [α] _ο = + 40.4 ° (СНС1 ₃ , c = 0.529) (direct crystallization, without prior chromatographic purification).

Example 3

In a 2-liter Erlenmeyer flask after loading 500 ml of a nutrient solution sterilized for 30 minutes at 121 ° C in an autoclave containing 3 wt.% Glucose, 1 wt.% Liquid obtained after soaking the corn kernels before swelling, 0.2 wt.% NaNO ₃ , 0.1 wt.% KN ₂ Ro _four , 0.2 wt.%

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TO ₂ NRA _four , 0.05 wt.% KC1, 0.05 wt.% Md8O _four -7H ₂ O and 0.002 wt.% Be8O _four -7H ₂ About (pH 6.0), half of the culture inoculum of the Akregd Shik osigaseik strain (CB8 13252), grown on beveled agar, was applied and shaken on a rotary shaker at 165 rpm for 72 hours at a rotational shaker. Then this preliminary culture was added to a 10-liter fermenter containing 9.5 liters of sterile medium of the same final composition as described above for the preliminary culture. In addition, prior to sterilization, another 0.5 ml of silicone oil and 0.5 ml of 8 mg (keto alcohol ethoxylate) were added to suppress pricing. At the end of the initial growth phase, which lasted for 6 hours at an elevated pressure of 0.7 bar, a temperature of 28 ° C, aeration at a flow rate of 5 l / min and a stirring speed of 350 rpm, a solution of 1.0 g of 7a-methylestr-4en was added. -3,17-dione in 15 ml of DMF. Then continued mixing and aeration. After 22 hours, the culture broth was collected and extracted twice with 4 liters of methyl isobutyl ketone for 4 hours. The combined organic phases were concentrated to dryness. The residue was almost completely dissolved in methanol. Silicone oil was filtered. Then it was concentrated and after chromatography on silica gel using a solvent gradient of dichloromethane / acetone, 0.78 g (74%) of 11a-hydroxy-7a-methyl-estr-4-ene-3,17-dione was isolated. After recrystallization from acetone / diisopropyl ether, 311 mg (29.6%) of white crystals were isolated with a melting point of 200 ° C and | α | _υ = + 52 ° (СНС1 ₃ c = 0.5905).

B. Chemical production method

Example 4. Obtaining 11B-fluoro-17B-hydroxy-7a-methyl-estr-4-en-3-one

a) 11 B-fluoro-7a-methylestr-4-ene-3.17-dione

To a solution of 13.08 g of 11a-hydroxy-7a-methyl-estr-4-ene-3,17-dione (obtained by microbiological synthesis proposed in the invention [section A]) in 250 ml of toluene and 18.2 ml of 1,8 diazabicyclo [5, 4,0] undec-7-ene at 0 ° C was added dropwise 11.5 ml of perfluorobutane-1 sulfonic acid fluoride. After 1 h, the mixture was neutralized with 2 molar hydrochloric acid, poured into water, extracted four times with ethyl acetate, washed with saturated sodium chloride solution, dried and concentrated in vacuo. After chromatography of the crude product on silica gel using a solvent gradient of hexane / ethyl acetate, 8.7 g of 11c-fluoro-7a-methylestr-4-ene-3,17-dione were obtained. Melting point 101.4 ° С, [a] _with = + 135.8 ° (СНС1 ₃ ).

b) 11v-fluoro-17v-hydroxy-7a-methyl-estr-4-en-3-one

A solution of 8.7 g of 11c-fluoro-7a-methyl-estr-4-en-3,17-dione in 148 ml of tetrahydrofuran was mixed dropwise at 0 ° C with 29.5 ml of 1-molar alumotretretbutoxyhydride of lithium and for 5.5 h stirred at 0 ° C. Then, diluted sulfuric acid was added at 0 ° C, after which the reaction solution was poured onto a mixture of ice and water, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using hexane / ethyl acetate. As a result, 5.8 g of 11B-fluoro-17B-hydroxy-7a-methyl estr-4-en-3-one with a melting point of 143-144 ° C were obtained, [a] _with = + 89.9 ° (СНС1 ₃ ).

Example 5. Obtaining 11B-fluoro-17B- (4-sulfamoylbenzoxy) -7a-methylestr-4-en-3-one

A solution of 500 mg of 11v-fluoro-17v-hydroxy-7a-methyl-estr-4-en-3-one in 7.5 ml of pyridine was mixed at room temperature with 750 mg of 4-sulfamoylbenzoic acid, 800 mg, dicyclohexylcarbodiimide and 125 mg η- The mixture was poured into sodium bicarbonate solution, extracted four times with dichloromethane, washed to a neutral state, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using dichloromethane / acetone. As a result, 302 mg of 11B-fluoro-17B- (4-sulfamoylbenzoxy) -7a-methylestr-4-en-3-one was obtained with a melting point of 232 ° C, [α] π = + 100.5 ° (СНС1 ₃ ).

Example 6. Obtaining 17a-ethynyl-11b-fluoro-17b-hydroxy-7a-methylestr-4-en-3-one

a) 11b-fluoro-3-methoxy-7a-methylestr-3,5-diene-17-one

A solution of 2 g of 11c-fluoro-7a-methyl-estr-4-ene-3,17-dione in 20 ml of 2,2-dimethoxypropane was mixed together with 200 mg of pyridinium tosylate for 6.5 hours at 80 ° C. Then it was diluted with ethyl acetate, washed with sodium bicarbonate solution and sodium chloride, dried over sodium sulfate and concentrated in vacuo. In this way, 2 g of crude 11B-fluoro-3-methoxy-7a-methylestra-3,5-diene-17-one was obtained.

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

A solution of 9.17 g of cerium chloride (111) in 60 ml of tetrahydrofuran was mixed drop by drop at 0 ° C with 74.2 ml of a solution of ethinyl magnesium bromide (0.5 molar solution in tetrahydrofuran) and stirred for 1 hour at 0 ° C. Then a solution of 2 g of crude 11B-fluoro-3-methoxy-7a-methylestr-3,5-dien17-one in 40 ml of tetrahydrofuran was added dropwise and stirring was continued for another 3.5 h at 0 ° C. For further processing, a saturated ammonium chloride solution was added, poured into water, extracted three times with ethyl acetate, washed with half-concentrated hydrochloric acid, sodium bicarbonate solution and sodium chloride, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using hexane / ethyl acetate. As a result, 1.15 g of pure 17a-ethynyl-11b-fluoro-17b-hydroxy-7a-methylestr-4-en-3-one was obtained with a melting point of 218-220 ° C, [α] π = + 19.2 ° (CHC1 ₃ ).

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

a) 3,3-ethanediyl-dioxy-17 a-ethynyl-11β-fluoroN-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 mixed together with 2.3 ml of trimethyl orthoformate and 30 mg of p-toluenesulfonic acid hydrate for 6.5 h at room temperature. The mixture was then poured into sodium bicarbonate solution, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using hexane / ethyl acetate. In this way, 205 mg of 3,3-ethanediol-dioxy-17α-ethynyl-11βfluoro-7a-methylestr-5 (10) -en-17β-ol was obtained.

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

A solution of 205 mg of 3,3-ethanediyl-dioxy-17α-ethynyl-11β-fluoro-7α-methylestr-5 (10) -ene-17β-ol in 27 ml of methanol and 3.6 ml of water was mixed together with 361 mg of oxalic acid for 24 h at room temperature. The mixture was then poured into sodium bicarbonate solution, extracted three times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using hexane / ethyl acetate. As a result, 95 mg of 17α-ethynyl-11β-fluoro-17β-hydroxy-7α-methyl estr-5 (10) -en-3-one was obtained with a melting point of 112-114 ° C.

Example 8. Obtaining 17α-ethynyl-11β-fluoro-7α-methylestr-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-methyl estr-4-ene-3,17-dione in 16.5 ml of acetonitrile was mixed together with 400 mg of copper (II) bromide for 6.5 hours at 25 ° C. Then it was diluted with ethyl acetate, washed with sodium bicarbonate solution and sodium chloride, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using hexane / acetone. In this way, 280 mg of pure 11β-fluoro-3-hydroxy-7α-methyl-estr-1,3,5 (10) -trien-17-one were obtained with a melting point of 185-186 ° C.

b) 17a-ethynyl-11β-fluoro-7 a-methylestra-1,3,5 (10) -trien-3, Ηβ-diol

A suspension of 2.03 g of cerium (III) chloride in 7.5 ml of tetrahydrofuran was mixed drop by drop at 0 ° C with 16.5 ml of a solution of ethinyl magnesium bromide (0.5 molar in tetrahydrofuran) and stirred for 0.5 h at 0 ° WITH. Then a solution of 280 mg of 11β-fluoro-3-hydroxy-7α-methyl-estr-1,3,5 (10) trien-17-one in 2.8 ml of tetrahydrofuran was added dropwise and stirring was continued for another 3.5 h at 0 ° s For further processing, a saturated solution of ammonium chloride was added, then poured into water, extracted four times with ethyl acetate, washed to neutrality, dried over sodium sulfate, concentrated in vacuo, and chromatographed on silica gel using hexane / ethyl acetate. As a result, 220 mg of 17α-ethynyl-11β-fluoro-7α-methyl estr-1,3,5 (10) -triene-3,17 β diol was obtained with a melting point of 115-117 ° C.

Claims (10)

CLAIM 1. Microbiological method of producing 7a-substituted 11 α-hydroxysteroids of the general formula 4, B in which K ⁷ is a P-P grouping, where P stands for C ₁ -C ₄ alkylene, and p stands for hydrogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ fluoroalkyl, and this group P-p through P is linked to the steroid skeleton, K ¹⁰ may be in the α or β position and is H, CH ₃ or CP _3, and K ¹³ is methyl or ethyl, consisting in the fact that 7a-substituted steroid of the general formula 3, A - 14 008147 in which K ⁷ , K ¹⁰ and K ¹³ have the above values, are hydroxylated and oxidized using a microorganism selected from the group consisting of Lsreg§111 sp., Veaiuepa sp., Slotega 11a sp., Spotto sp., Narozrogea 11a . and KY / oriz sp. 2. The method according to p. oriz z1o1ot £ er. 3. A microbiological method for the preparation of 7a-substituted 11a-hydroxysteroids of the general formula 4, B in which K ⁷ is a group P ^, where P stands for C ₁ -C ₄ alkylene, and C stands for hydrogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ fluoroalkyl, and this group P-9 is linked through P with the steroid carcass, K ¹⁰ may be in the α or β position and is H, CH ₃ or CP _3, and K ¹³ is methyl or ethyl, consisting in the fact that 7a-substituted steroid of general formula 3, A in which K ⁷ , K ¹⁰ and K ¹³ have the above values, in the first stage of the microbiological method using an appropriate microorganism selected from the group , including LzregdSiz sp., Veaiuepa sp., Clerk-e11a sp., O1otegade 11a., Spotto sp., Meyaggyy7sht., KG / OGP sp., KY / Ors sp. and Hygienic sp., hydroxylate at position 11α to form 7a-substituted 11α- in which K ⁷ , K ¹⁰ and K ¹³ are as defined above, then formed by a 7a-substituted 11α-15 008147 hydroxysteroid of the general formula C in the second stage of the microbiological method using another microorganism selected from the group including Vaschiz sp., Muscle pulp sp., ^ SagSa sp. and Reazidotopaz sp., is oxidized to form a 7α-substituted steroid of general formula 4, B. 4. A method of making a difference. Rez, Opotto stdikPa, Metiaggli / 1it AshzorPaye, the number of the health of the Red Cross, you / oriz ogu / ae, you / oriz z! 5. The method according to claims 3 and 4, characterized in that the microorganism used in the second stage is selected from the group consisting of Vasromacchia, Vaschis zyrAlepisis, Musobacter péoigit, Musobactivitis ztedtpz, Arthurus sapphire, Sausagetrophicus zedtarz, Arthurus sapphire, Sausagetrophicus zedtprz, Arthurus Saigonusa, Sausagetrophilustixustidaum, Arthropyracidum, Arthropyracidae, Arthoriacetumus, Arthritisustixus, Arthritisceroceaeisus, Arthritisus, Arthritisceraceaeceaeceaeceaeusis, Herculosa Sthyraxinus. GeResSa, SagSaGygoregRps1a, SagSa ZaTishoS1og and Rezeidotopaz Izloz1ogopt 6. Microbiological method of producing 7α-substituted 11 α-hydroxysteroids of general formula 4, B in which B ⁷ is a P-0 grouping, where P is C ₁ -C ₄ alkylene, and O is hydrogen, C ₁ -C ₄ alkyl or C ₁ -C ₄ fluoroalkyl, and this group P-0 is linked through P to the steroid carcass, B ¹⁰ is H, CH ₃ or CE ₃ and B ¹³ is methyl or ethyl, consisting in the fact that 7α-substituted steroids of the general formula Ώ in which B ⁷ , B ¹⁰ and B ¹³ are as defined above, are hydroxylated using a microorganism selected from the group consisting of Weiuyera sp., Siguyyla sp., Oleber sp., Oteotera sp., Opotosh sp., Narozrogero st. , Health certificate number and 8upserIa1az1git sp. 7. A method according to claim 6, characterized in that the microorganism is selected from the group consisting Veaiuepa azz1apa, Sigui1apa 1ipa1a, O1ege11a / eae, O1otege11a stdikPa, O1otege11a Gizago1dez, Opotosha sshdi1a! A, Nar1ozroge11a IezregeShsa, NePsoz1u1it rShGogtae, №dgozroga zryaepsa and 8upserya1az1git gasetozit. 8. Microbiological method according to one of claims 1 to 7, characterized in that B ⁷ is CH3. 9. Microbiological method according to one of claims 1 to 8, characterized in that B ¹⁰ is N. 10. Microbiological method according to one of claims 1 to 9, characterized in that B ¹³ is CH3.