RU2261865C2 - METHOD FOR PREPARING 3-KETO-7α-ALKOXYCARBONYL-SUBTITUTED Δ4,5-STEROID, METHODS FOR PREPARING INTERMEDIATE COMPOUNDS, INTERMEDIATE COMPOUNDS - Google Patents

Abstract

FIELD: organic chemistry, steroids, chemical technology.

SUBSTANCE: invention describes a method for preparing 3-keto-7α-alkoxycarbonyl-substituted △4,5-steroid of the formula (I):

wherein

is taken among

or

R³ means hydrogen atom (H), lower alkyl, lower alkoxy-group or cyano-group (CN); R²¹ means hydrogen atom (H) or alkyl; R²⁶ means (C₁-C₄)-alkyl; R⁸ and R⁹ form in common heterocyclic ring system. Method involves interaction of an alkylating agent with 4,5-dihydro-5,7-lactone steroid of the formula (II):

wherein R¹⁸ means (C₁-C₄)-alkyl or R¹⁸O-group taken in common form O,O-oxyalkylene bridge or keto-group and

R³, R⁸ and R⁹ have above given values in the presence of a base. Compounds of the formula (I) are used as intermediate compounds in improved methods for synthesis of epoxymexerone.

EFFECT: improved preparing method.

56 cl, 42 tbl, 30 sch, 5 dwg, 89 ex

Description

BACKGROUND OF THE INVENTION

The present invention relates to new methods for producing 9,11-epoxysteroid derivatives, in particular compounds of the series of 20-spiroxanes and their analogues; to new intermediates used to prepare steroid compounds; and methods for producing these new intermediates. More specifically, the invention relates to new and improved methods for producing methylhydro 9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone (also called eplerenone or epoxymexrenone) (γ monomethyl ether -lactone 9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylic acid).

Methods for producing compounds of the 20-spiroxane series are described in US Pat. No. 4,559,332. Compounds produced by the method described in patent 4559332, have an open oxygen-containing ring E of the General formula:

Where

-A-A- represents the group —CH ₂ —CH ₂ - or —CH = CH—;

R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical;

-B-B- represents a group -CH ₂ -CH ₂ - or an α- or β-oriented group;

R ⁶ and R ⁷ represent hydrogen;

X represents two hydrogen atoms or oxo;

Y ¹ and Y ² together represent an oxygen bridge —O—, or

Y ¹ represents hydroxy, and

Y ² is hydroxy, lower alkoxy, or, if X is H ₂ , also lower alkanoyloxy;

and salts of such compounds in which X is oxo and Y ² is hydroxy, that is, salts of the corresponding 17β-hydroxy-21-carboxylic acids.

US Pat. No. 4,559,332 describes a number of methods for preparing epoxymexrenone and structurally similar compounds of formula IA. The emergence of new and wider clinical possibilities for the use of epoxymexrenone leads to the need to improve the methods for producing it and other steroids with a similar structure.

SUMMARY OF THE INVENTION

The main objective of the present invention is the development of improved methods for producing epoxymexrenone, other 20-spiroxanes and other steroids having common structural features. Some of the specific objectives of the present invention are: the development of an improved process for the preparation of products of formula IA and other structurally similar compounds in high yield; the development of such a method, which involves the implementation of a minimum of stages of selection; and the development of such a method that can be implemented with reasonable capital costs and with reasonable costs associated with the conversion.

In accordance with this, the present invention relates to a number of schemes for the synthesis of epoxymexrenone; intermediates that can be used to produce epoxymexrenone; and the synthesis of such new intermediates.

New synthesis schemes are detailed in the description of preferred embodiments of the invention. Part of these new intermediates of the present invention are presented immediately below.

The compound of formula IV corresponds to the structure

Where:

-A-A- represents the group —CHR ⁴ —CHR ⁵ - or —CR ⁴ = CR ⁵ -;

R ³ , R ⁴ and R ^{5 are} independently selected from the group consisting of hydrogen, halogen, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;

R ¹ represents an alpha oriented lower alkoxy carbonyl or hydroxycarbonyl radical;

R ² represents an 11α-leaving group, the cleavage of which is effective for introducing a double bond between 9- and 11-carbon atoms;

-B-B represents a group -CHR ⁶ -CHR ⁷ -, or an alpha or beta oriented group:

where R ⁶ and R ^{7 are} independently selected from the group consisting of hydrogen, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and

R ⁸ and R ^{9 are} independently selected from the group consisting of hydrogen, hydroxy, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ form a carbocyclic or heterocyclic ring structure fused to the pentacyclic ring D.

The compound of formula IVA corresponds to formula IV, where R ⁸ and R ⁹ together with the ring carbon atom to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula IVB corresponds to formula IV, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas IVC, IVD and IVE, respectively, correspond to any of formulas IV, IVA or IVB, where each of -A-A- and -B-B represents -CH ₂ -CH ₂ -, R ³ represents hydrogen and R ¹ represents alkoxycarbonyl, preferably methoxycarbonyl. Compounds of Formula IV can be prepared by reacting a lower alkylsulfonylating or acylating reagent or a halide forming agent with the corresponding compound of Formula V.

The compound of formula V corresponds to the structure:

where: -A-A-, -B-B-, R ¹ , R ³ , R ⁸ and R ^{9 are} defined in formula IV.

The compound of formula VA corresponds to formula V, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula VB corresponds to formula V, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas VC, VD and VE, respectively, correspond to any of formulas V, VA or VB, where each of -A-A- and -B-B represents -CH ₂ -CH ₂ -, R ³ represents hydrogen and R ¹ represents hydrogen alkoxycarbonyl, preferably methoxycarbonyl. Compounds covered by formula V can be prepared by reacting an alkali metal alkoxide with a corresponding compound of formula VI.

The compound of formula VI corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula IV.

The compound of formula VIA corresponds to formula VI, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula VIB corresponds to formula VI, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

Compounds of formulas VIC, VID and VIE, respectively, correspond to any of formulas VI, VIA or VIB, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ -, and R ³ is hydrogen. Compounds of formulas VI, VIA, VIB and VIC are prepared by hydrolysis of a compound corresponding to formulas VII, VIIA, VIIB or VIIC, respectively.

The compound of formula VII corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula VIIA corresponds to formula VII, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula VII B corresponds to formula VII, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas VIIC, VIID and VIIE, respectively, correspond to any of formulas VII, VIIA or VIIB, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ -, and R ³ is hydrogen. The compound covered by formula VII can be obtained by cyanidation of the compounds covered by formula VIII.

The compound of formula VIII corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined above in formula IV.

The compound of formula VIIIA corresponds to formula VIII, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula VIII B corresponds to formula VIII, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas VIIIC, VIIID and VIIIE, respectively, correspond to any of formulas VIII, VIIIA or VIIIB, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ - and R ³ is hydrogen. Compounds of Formula VIII are prepared by oxidizing a substrate containing a compound of Formula XXX, as described below, by fermentation to introduce the 11-hydroxy group into the substrate in the α orientation. The compound of formula IX corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined above for formula IV, and R ^{1 is} defined for formula V.

The compound of formula IXA corresponds to formula IX, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula IXB corresponds to formula IX, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIII:

The compounds of formulas IXC, IXD and IXE, respectively, correspond to any of formulas IX, IXA or IXB, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ - and R ³ is hydrogen. Compounds covered by formula IX can be prepared by conversion of the corresponding compound of formula X.

The compound of formula XIV corresponds to the structure;

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined above in formula IV.

The compound of formula XIVA corresponds to formula XIV, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula XIVB corresponds to formula XIV, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIII:

The compounds of formulas XIVC, XIVD and XIVE, respectively, correspond to any of formulas XIV, XIVA or XIVB, where each of —A — A— and —B — B— represents —CH ₂ —CH ₂ - and R ³ represents hydrogen. Compounds covered by formula XIV can be prepared by hydrolysis of the corresponding compound of formula XV.

The compound of formula XV corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula XVA corresponds to formula XV, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form a structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula XVB corresponds to formula XV, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIII:

The compounds of formulas XVC, XVD and XVE, respectively, correspond to any of formulas XV, XVA or XVB, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ - and R ³ represents hydrogen. Compounds of Formula XV may be prepared by cyanidation of the corresponding compound of Formula XVI.

The compound of formula XXI corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula XXIA corresponds to formula XXI, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula XXIB corresponds to formula XXI, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas XXIC, XXID and XXIE, respectively, correspond to any of formulas XXI, XXIA or XXIB, where each of —A — A— and —B — B— represent —CH ₂ —CH ₂ -, and R ³ represents hydrogen. Compounds covered by formula XXI can be obtained by hydrolysis of the corresponding compounds covered by formula XXII.

The compound of formula XXII corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula XXIIA corresponds to formula XXII, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula XXIIB corresponds to formula XXII, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas XXIIC, XXIID and XXIIE, respectively, correspond to any of formulas XXII, XXIIA or XXIIB, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen. Compounds covered by formula XXII can be prepared by cyanidation of the corresponding compound of formula XXIII.

The compound of formula XXIII corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula XXIIIA corresponds to formula XXIII, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula XXIIIB corresponds to formula XXIII, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas XXIIIC, XXIIID and XXIIIE, respectively, correspond to any of formulas XXIII, XXIIIA or XXIIIB, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ - and R ³ represents hydrogen. Compounds covered by formula XXIII can be prepared by oxidizing a compound of formula XXIV, as described below.

The compound of formula XXVI corresponds to the structure:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula XXVIA corresponds to formula XXVI, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ - and R ³ is hydrogen. Compounds encompassed by formula XXVI can be prepared by oxidizing a compound of formula XXVII.

The compound of formula XXV corresponds to the structure:

Where; -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above for formula IV.

The compound of formula XXVA corresponds to formula XXV, where each of —A — A— and —B — B— represents a —CH ₂ —CH ₂ - group, and R ³ represents hydrogen. Compounds covered by formula XXV can be prepared by cyanidation of a compound of formula XXVI.

The compound of formula 104 corresponds to the structure:

where: -A-A-, -B-B- and R ^{3 are as} defined above for formula IV, and R ¹¹ is C ₁ -C ₄ alkyl.

The compound of formula 104A corresponds to formula 104, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ -, and R ³ is hydrogen. Compounds of Formula 104 can be prepared by thermal decomposition of a compound of Formula 103.

The compound of formula 103 corresponds to the structure:

where: -A-A-, -B-B-, R ³ and R ^{11 are as} defined above for formula 104, and R ¹² is C ₁ -C ₄ alkyl.

The compound of formula 103A corresponds to formula 103, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ -, and R ³ is hydrogen. Compounds encompassed by formula 103 can be prepared by reacting the corresponding compound of formula 102 with dialkyl malonate in the presence of a base such as an alkali metal alkoxide.

The compound of formula 102 corresponds to the structure;

where: -A-A-, -B-B-, R ³ and R ^{11 are} defined above for formula 104.

The compound of formula 102A corresponds to formula 102, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ -, and R ³ is hydrogen. Compounds covered by formula 102 can be prepared by reacting the corresponding compound of formula 101 with a trialkylsulfonium compound in the presence of a base.

The compound of formula 101 corresponds to the structure:

where: -A-A-, -B-B-, R ³ and R ^{11 are} defined above for formula 104.

The compound of formula 101A corresponds to formula 101, where each of —A — A— and —B — B— is —CH ₂ —CH ₂ -, and R ³ is hydrogen. Compounds covered by formula 101 can be prepared by reacting 11α-hydroxyandrosten-3,17-dione or another compound of formula XXXVI with trialkyl ortho-formate in the presence of an acid.

The compound of formula XL corresponds to the formula;

where -E-E- is selected from:

and

R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, alkyl, halogen, nitro and cyano; R ^{24 is} selected from hydrogen and lower alkyl; R ⁸⁰ and R ^{90 are} independently selected from keto and substituents, which may be R ⁸ and R ⁹ (as defined above with respect to formula IV); and -A-A-, -B-B- and R ^{3 are} defined for formula IV.

The compound of formula XLA corresponds to formula XL, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula XLB corresponds to formula XLA, where -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII. The compound of formula XLC corresponds to formula XLB, where -E-E- corresponds to formula XLV. Compound XLD corresponds to formula XLB, where -E-E- corresponds to formula XLVII.

The compound of formula XLE corresponds to formula XL, where R ⁸⁰ and R ⁹⁰ together with the ring carbon to which they are attached represent keto or:

where X, Y ¹ , Y ² and C (17) are defined above or

The compounds of formula XLIE correspond to formula XL, where R ⁸⁰ and R ⁹⁰ together form a keto.

Compounds of the formulas XLF, XLG, XLH, XLJ, XLM and XLN correspond to the formulas XL, XLA, XLB, XLC, XLD and XLE, respectively, in which -A-A-, -B-B- and R ^{3 are} defined above.

The compound of formula XLI corresponds to the formula:

where -E-E- is selected from:

and

R ¹⁸ represents C ₁ -C ₄ alkyl, or R ¹⁸ O groups — together form an O, O-hydroxyalkylene bridge; R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, alkyl, halogen, nitro and cyano; R ^{24 is} selected from hydrogen and lower alkyl; R ⁸⁰ and R ^{90 are} independently selected from keto and substituents, which may be R ⁸ and R ⁹ ; and -A-A-, -B-B- and R ^{3 are} defined in formula IV.

The compound of formula XLIA corresponds to formula XLI, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula XLIB corresponds to formula XLIA, where -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

The compound of formula XLIC corresponds to formula XLI, where R ⁸⁰ and R ⁹⁰ together with the ring carbon to which they are attached represent keto or:

where: X, Y ¹ , Y ² and C (17) are defined above.

Compounds of formula XLID correspond to formula XLI, where substituent XXXIV corresponds to structure XXXIII

The compounds of formula XLIE correspond to formula XL, where R ⁸⁰ and R ⁹⁰ together form a keto.

Compounds of formulas XLIF, XLIG, XLIH, XLIJ, XLIM and XLIN correspond to formulas XLI, XLIA, XLIB, XLIC, XLID and XLIE, respectively, where -A-A-, -B-B- and R ^{3 are} defined above. Compounds encompassed by Formula XLI are prepared by hydrolysis of the corresponding compounds of Formula XL shown below.

The compound of formula XLII corresponds to the formula:

where -E-E- is selected from:

and

R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, alkyl, halogen, nitro and cyano; R ^{24 is} selected from hydrogen and lower alkyl; R ⁸⁰ and R ^{90 are} independently selected from keto and substituents, which may be R ⁸ and R ⁹ , and —A — A—, —B — B— and R ^{3 are} defined in formula IV.

The compound of formula XLIIA correspond to formula XLII, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula XLIIB correspond to formula XLIIA, where -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

The compound of formula XLIIC corresponds to formula XLII, where R ⁸⁰ and R ⁹⁰ together with the ring carbon to which they are attached represent keto or:

where X, Y ¹ , Y ² and C (17) are defined above.

Compounds of formula XLIID correspond to formula XLII, where substituent XXXIV corresponds to structure XXXIII

The compounds of formula XLIIE correspond to formula XLII, where R ⁸⁰ and R ⁹⁰ together form a keto. The compounds of formulas XLIIF, XLIIG, XLIIH, XLIIJ, XLIIM and XLIIN correspond to formulas XLII, XLIIA, XLIIB, XLIIC, XLIID and XLIIE, respectively, where -A-A- and -B-B- represent -CH ₂ -CH ₂ -, and R ³ represents hydrogen. Compounds of Formula XLII are prepared by deprotection of the corresponding compound of Formula XLI.

The compound of formula XLIX correspond to the structure:

where -E-E- is defined for formula XL, and -A-A-, -B-B-, R ¹ , R ³ , R ⁸ and R ^{9 are} defined in formula IV.

The compound of formula XLIXA corresponds to formula XLIX, where R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula XLIXB corresponds to formula XLIX, where R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of formulas XLIXC, XLIXD, XLIXE, respectively, correspond to any of formulas XLIX, XLIXA or XLIXB, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, R ³ represents hydrogen, and R ¹ represents alkoxycarbonyl, preferably methoxycarbonyl. Compounds covered by formula XLIX can be prepared by reacting an alcohol or aqueous solvent with a corresponding compound of formula VI in the presence of a suitable base.

The compound of formula A203 corresponds to the structure:

where -E-E- is selected from:

and

R ^{18 is} selected from C ₁ -C ₄ alkyl; R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, alkyl, halogen, nitro and cyano; R ^{24 is} selected from hydrogen and lower alkyl; a-A-A-, -B-B- and R ^{3 are} defined for formula IV.

The compound of formula A203A corresponds to formula A203, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula A203B corresponds to formula A203A, where -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

The compounds of formulas A203C, A203D and A203E, respectively, correspond to formulas A203, A203A and A203B, where each of —A — A— and —B — B— represents —CH ₂ —CH ₂ -, and R ³ represents hydrogen. Compounds of Formula A203 are prepared by reducing a compound of Formula A202 below.

The compound of formula A204 corresponds to the structure:

where R ¹⁹ represents C ₁ -C ₄ -alkyl and -E-E-, -A-A-, -B-B- and R ^{3 are} defined for formula 203.

The compound of formula A204A corresponds to formula A204, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula A204B corresponds to formula A204A, where -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

The compounds of formulas A204C, A204D and A204E, respectively, correspond to formulas A204, A204A and A204B, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen. Compounds of Formula A204 are prepared by hydrolysis of the corresponding compounds of Formula A203.

The compound of formula A205 corresponds to the structure:

where R ²⁰ represents C ₁ -C ₄ -alkyl, a -E-E-, R ¹⁹ , -A-A-, -B-B- and R ^{3 are} defined for formula 204.

The compound of formula A205A corresponds to formula A205, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula A205B corresponds to formula A205A, where -E-E- corresponds to formulas XLIII, XLIV, XLV or XLVII.

The compounds of formulas A205C, A205D and A205E, respectively, correspond to formulas A205, A205A and A205B, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen. Compounds of Formula A205 can be prepared by reacting the corresponding compound of Formula A204 with an alcohol and an acid.

The compound of formula A206 corresponds to the structure:

where R ¹⁹ , R ²⁰ , -E-E-, -A-A-, -BB- and R ^{3 are} defined for formula 205.

The compound of formula A206A corresponds to formula A206, wherein R ²¹ , R ^22, and R ^{23 are} independently selected from hydrogen, halogen, and lower alkyl.

The compound of formula A206B corresponds to formula A206A, where -E-E- corresponds to formulas XLIII, XLIV, XLV or XLVII.

The compounds of formulas A206C, A206D and A206E, respectively, correspond to formulas A206, A206A and A206B, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen. Compounds of Formula A206 can be prepared by reacting the corresponding compound of Formula A205 with a trialkylsulfonium halide.

The compound of formula A207 corresponds to the structure:

where R ²⁵ represents C ₁ -C ₄ alkyl and -E-E-, R ¹⁹ , R ²⁰ , -A-A-, -B-B- and R ^{3 are} defined for formula A205.

The compound of formula A207A corresponds to formula A207, where R ²¹ , R ²² and R ^{23 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula A207B corresponds to formula A207A, where -E-E- corresponds to formulas XLIII, XLIV, XLV or XLVII.

The compounds of formulas A207C, A207D and A207E, respectively, correspond to formulas A207, A207A and A207B, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ - and R ³ represents hydrogen. Compounds of Formula A207 can be prepared by reacting the corresponding compound of Formula A206 with dialkyl malonate.

The compound of formula A208 corresponds to the structure:

where -E-E-, R ⁸⁰ and R ^{90 are} defined for formula XLII; -A-A-, -B-B- and R ^{3 are} defined in formula 104; and R ¹⁹ , R ²⁰ , -A-A-, -B-B- and R ^{3 are} defined for formula 205.

The compound of formula A208A corresponds to formula A208, where R ²¹ and R ^{22 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula A208B corresponds to formula A208A, where -E-E- corresponds to formulas XLIII, XLIV, XLV or XLVII.

The compound of formula A208C corresponds to formula A208, where R ⁸⁰ and R ⁹⁰ together with the ring carbon to which they are attached represent keto or:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula 208D corresponds to formula 208C, in which Deputy XXXIV corresponds to structure XXXIII:

The compounds of formulas A208E, A208F, A208G, A208H and A208J, respectively, correspond to formulas A208, A208A, A208B, A208C and A208D, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ , and R ³ represents hydrogen. Compounds covered by Formula A208 can be prepared by thermal decomposition of the corresponding compounds of Formula A207.

The compound of formula A209 corresponds to the structure:

where R ⁸⁰ and R ^{90 are} defined for formula XLI, and -E-E-, -A-A-, -B-B- and R ^{3 are} defined in formula 205.

The compound of formula A209A corresponds to formula A209, where R ²¹ and R ^{22 are} independently selected from hydrogen, halogen and lower alkyl.

The compound of formula A209B corresponds to formula A209A, where -E-E- corresponds to formulas XLIII, XLIV, XLV or XLVII.

The compound of formula A209C corresponds to formula A209B, where -E-E- corresponds to formula XLIV.

The compound of formula A209D corresponds to formula A208, where R ⁸⁰ and R ⁹⁰ together with the ring carbon to which they are attached represent keto or:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula 209E corresponds to formula 209D, where the Deputy XXXIV corresponds to structure XXXIII:

The compounds of formulas A209F, A209G, A209H, A209J, A209L and A209M, respectively, correspond to formulas A209, A209A, A209B, A209C, A208D and A209E, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen. Compounds covered by Formula A209 can be prepared by hydrolysis of the corresponding compound of Formula A208.

The compound of formula A210 corresponds to the structure;

where R ⁸⁰ and R ^{90 are} defined for formula XLI, and the substituents -A-A-, -B-B- and R ^{3 are} defined for formula IV.

The compound of formula A210A corresponds to formula A210, where R ⁸⁰ and R ⁹⁰ together with the ring carbon to which they are attached represent keto or:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula A210B corresponds to the formula A210A, where the substituent XXXIV corresponds to structure XXXIII:

The compound of formula A210C corresponds to formula A210A, where R ⁸⁰ and R ⁹⁰ together form a keto.

.Compounds of the formulas A210D, A210E, A210F and A210G, respectively, correspond to the formulas A210, A210A, A210B and A210C, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen . Compounds of Formula 210 can be prepared by epoxidizing a compound of Formula 209 in which —E — E— represents

.

The compound of formula A211 corresponds to the formula:

where -A-A-, -B-B- and R ^{3 are} defined above.

The compound of formula A211A corresponds to formula A211, where R ⁸⁰ and R ⁹⁰ together form a keto or:

where X, Y ¹ , Y ² and C (17) are defined above.

The compound of formula A211B corresponds to the formula A211A, in which Deputy XXXIV corresponds to structure XXXIII:

The compound of formula A211C corresponds to formula A211A, where R ⁸⁰ and R ⁹⁰ together form a keto.

. Соединения формулы А211 могут быть преобразованы в соединения формулы I способом, описанным ниже.The compounds of the formulas A211D, A211E, A211F and A211G, respectively, correspond to the formulas A211, A211A, A211B and A211C, where each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -, and R ³ represents hydrogen . Compounds covered by the formula A211 can be obtained by oxidation of the corresponding compound of the formula A210, or by epoxidation of the corresponding compound of the formula A209, where -E-E- represents

. Compounds of formula A211 may be converted to compounds of formula I by the method described below.

The compound of formula L corresponds to the structure:

where R ¹¹ represents C ₁ -C ₄ alkyl, and -A-A-, -B-B-, R ¹ , R ² , R ³ , R ⁸ and R ^{9 are as} defined above.

The compounds of formula LA correspond to formula L, where R ⁸ and R ⁹ together with the carbon to which they are attached represent:

where X, Y ¹ and Y ^{2 are} defined above.

The compounds of formula LB correspond to formula L, where R ⁸ and R ⁹ correspond to structure XXXIII

Compounds of formulas LC, LD, LE correspond to formulas L, LA and LB, respectively, where each of —A — A— and —B — B— represents —CH ₂ —CH ₂ -, and R ³ represents hydrogen.

Based on the description of the specific reaction schemes presented below, it will be possible to determine which of these compounds are most suitable for a given reaction scheme. The compounds of the present invention can be used as intermediates for the preparation of epoxymexrenone and other steroids.

Other objectives and features of the present invention are partly obvious, and partly described below.

Brief Description of the Drawings

Figure 1 presents a diagram of a method for the biological conversion of canrenone or a derivative of canrenone into the corresponding 11α-hydroxy compound;

figure 2 presents a diagram of a preferred method for the biological conversion / 11-α-hydroxylation of canrenone and derivatives of canrenone;

figure 3 presents a diagram of a particularly preferred method of biological conversion / 11-α-hydroxylation of canrenone and derivatives of canrenone;

figure 4 presents the particle size distribution of canrenone, obtained in accordance with the method illustrated in figure 2; and

figure 5 shows the particle size distribution of canrenone sterilized in a fermenter for transformation by the method illustrated in figure 3.

The corresponding item numbers in the drawings indicate the corresponding parts of these drawings.

Description of preferred embodiments of the present invention

In accordance with the present invention have been developed various new schemes of methods for producing epoxymexrenone and other compounds corresponding to the formula I:

Where:

-A-A- represents the group —CHR ⁴ —CHR ⁵ - or —CR ⁴ = CR ⁵ -;

R ³ , R ⁴ and R ^{5 are} independently selected from the group consisting of hydrogen, halogen, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;

R ¹ represents an α-oriented lower alkoxycarbonyl or hydroxyalkyl radical; and

-B-B represents a group -CHR ⁶ -CHR ⁷ - or an alpha or beta oriented group:

where R ⁶ and R ^{7 are} independently selected from the group consisting of hydrogen, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and

R ⁸ and R ^{9 are} independently selected from the group consisting of hydrogen, hydroxy, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; either R ⁸ and R ⁹ taken together represent a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ taken together with R ⁶ or R ⁷ represent a carbocyclic or heterocyclic ring structure fused to the pentacyclic ring D.

Unless otherwise specified, organic radicals, referred to herein as "lower", contain a maximum of 7, and preferably from 1 to 4 carbon atoms.

The lower alkoxycarbonyl radical is preferably a radical derived from an alkyl radical having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl; particularly preferred are methoxycarbonyl, ethoxycarbonyl and isopropoxycarbonyl. The lower alkoxy radical is preferably a radical derived from one of the aforementioned C ₁ -C ₄ alkyl radicals, especially from a primary C ₁ -C ₄ alkyl radical; particularly preferred is methoxy. The lower alkanoyl radical is, preferably, a radical derived from direct alkyl having from 1 to 7 carbon atoms; and particularly preferred are formyl and acetyl.

The methylene bridge in the 15.16 position is preferably β-oriented.

A preferred class of compounds that can be prepared by the methods of the present invention are the 20-spiroxane compounds described in US Pat. No. 4,559,332, i.e. compounds corresponding to formula IA

Where:

-A-A- represents the group —CH ₂ —CH ₂ - or —CH = CH—;

—B — B— represents a —CH ₂ —CH ₂ - group, or an alpha or beta oriented group of formula IIIA:

R ¹ represents an alpha oriented lower alkoxycarbonyl or hydroxycarbonyl radical;

X represents two hydrogen atoms, oxo or = S;

Y ¹ and Y ² taken together represent an oxygen bridge —O— or

Y ¹ represents hydroxy, and

Y ² represents hydroxy, lower alkoxy, or, if X represents H ₂ , then also lower alkanoyloxy.

Preferably, the 20-spiroxane compounds prepared by the new methods of the present invention are compounds of formula I, wherein Y ¹ and Y ² taken together represent an oxygen bridge —O—.

Particularly preferred compounds of formula I are compounds in which X is oxo. Of the 20-spiroxane compounds of formula 1A, where X is oxo, those compounds in which Y ¹ together with Y ² represent the oxygen bridge —O— are most preferred.

As already mentioned, 17β-hydroxy-21-carboxylic acid can also be obtained in the form of its salts. In particular, metal and ammonium salts such as alkali metal and alkaline earth metal salts, for example sodium, calcium, magnesium salts, and preferably potassium salts are contemplated; and ammonium salts derived from ammonia or a suitable, preferably physiologically acceptable, organic nitrogen-containing base. Not only amines are considered as bases, for example lower alkylamines (such as triethylamine), hydroxy (lower) alkylamines (such as 2-hydroxyethylamine, di- (2-hydroxyethyl) amine or tri- (2-hydroxyethyl) amine), cycloalkylamines ( such as dicyclohexylamine), or benzylamines (such as benzylamine and N, N'-dibenzylethylenediamine), but also nitrogen-containing heterocyclic compounds, for example aromatic compounds (such as pyridine or quinoline), or compounds having at least partially saturated heterocyclic ring ( the same e like N-ethylpiperidine, morpholine, piperazine, or N, N'-dimethylpiperazine).

Preferred compounds are also alkali metal salts, especially potassium salts of compounds of formula IA, wherein R ¹ is alkoxycarbonyl, X is oxo, and each of Y ¹ and Y ² is hydroxy.

Particularly preferred compounds of the formula I and IA are, for example, the following compounds:

9α, 11α-epoxy-7α-methoxycarbonyl-20-spirox-4-en-3,21-dione;

9α, 11α-epoxy-7α-ethoxycarbonyl-20-spirox-4-en-3,21-dione;

9α, 11α-epoxy-7α-isopropoxycarbonyl-20-spirox-4-en-3,21-dione

and 1,2-dehydro analogs of each of these compounds;

9α, 11α-epoxy-6α, 7α-methylene-20-spirox-4-en-3,21-dione;

9α, 11α-epoxy-6β, 7β-methylene-20-spirox-4-en-3,21-dione;

9α, 11α-epoxy-6β, 7β, 15β, 16β-bismethylene-20-spirox-4-en-3,21-dione

and 1,2-dehydro analogs of each of these compounds;

9α, 11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-en-21-carboxylic acid;

9α, 11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-en-21-carboxylic acid;

9α, 11α-epoxy-7α-isopropoxycarbonyl-17β-hydroxy-3-oxo-pregn-4-en-21-carboxylic acid;

9α, 11α-epoxy-17β-hydroxy-6α, 7α-methylene-3-oxo-pregn-4-en-21-carboxylic acid;

9α, 11α-epoxy-17β-hydroxy-6β, 7β-methylene-3-oxo-pregn-4-en-21-carboxylic acid,

9α, 11α-epoxy-17β-hydroxy-6β, 7β, 15β, 16β-bismethylene-3-oxo-pregn-4-en-21-carboxylic acid,

and alkali metal salts, especially potassium or ammonium salts of each of these acids, as well as the corresponding 1,2-dehydro analogs of each of the aforementioned carboxylic acids or their salts;

9α, 11α-epoxy-15β, 16β-methylene-3,21-dioxo-20-spirox-4-ene-7α-carboxylic acid methyl, ethyl and isopropyl ester;

9α, 11α-epoxy-15β, 16β-methylene-3,21-dioxo-20-spirox-1,4-diene-7α-carboxylic acid methyl, ethyl and isopropyl ester;

9α, 11α-epoxy-3-oxo-20-spirox-4-en-7α-carboxylic acid methyl, ethyl and isopropyl ester;

9α, 11α-epoxy-6β, 6β-methylene-20-spirox-4-en-3-one;

9α, 11α-epoxy-6β, 7β, 15β, 16β-bismethylene-20-spirox-4-en-3-one;

9α, 11α-epoxy-17β-hydroxy-17α (3-hydroxypropyl) -3-oxo-adrost-4-en-7α-carboxylic acid methyl, ethyl and isopropyl ester;

9α, 11α-epoxy-17β-hydroxy-17α- (3-hydroxypropyl) -6α, 7α-methylene-androst-4-en-3-one,

9α, 11α-epoxy-17β-hydroxy-17α- (3-hydroxypropyl) -6β, 7β-methylene-androst-4-en-3-one,

9α, 11α-epoxy-17β-hydroxy-17α- (3-hydroxypropyl) -6β, 7β, -15β, 16β-bismethylene-androst-4-en-3-one,

including 17α- (3-acetoxypropyl) - and 17α- (3-formyloxy-propyl) analogues of the aforementioned androstane compounds,

as well as 1,2-dehydro analogs of all types of the aforementioned compounds androst-4-en-3-one and 20-spirox-4-en-3-one.

The chemical names of the compounds of formulas I and IA and analog compounds having the same characteristic structural features are given in accordance with generally accepted nomenclature, namely: the names for compounds in which Y ¹ together with Y ² represents —O come from 20-spiroxane (for example, a compound of formula IA where X is oxo and Y ¹ taken together with Y ² is —O is called “20-spiroxan-21-one”); the name for compounds in which each of Y ¹ and Y ² represents hydroxy and X represents oxo, is derived from 17β-hydroxy-17α-pregnen-21-carboxylic acid; and the name for compounds in which each of Y ¹ and Y ² represents hydroxy and X represents two hydrogen atoms, comes from 17β-hydroxy-17α- (3-hydroxypropyl) -androstane. Since cyclic and open-chain forms, i.e., lactones and 17β-hydroxy-21-carboxylic acids and their salts, respectively, are so close to each other that the latter can only be considered as the hydrated form of the former, it should be noted that in the preceding and following description, unless otherwise indicated, these forms, both in the final products of formula I, and in the starting and intermediate compounds of a similar structure, are referred to together in all cases.

In accordance with the present invention, several separate schemes have been developed for preparing compounds of formula I in high yield and at moderate material cost. Each of these synthesis schemes involves the preparation of a series of intermediate compounds. A number of these intermediates are new compounds, and methods for preparing these intermediates are new methods.

Scheme 1 (using starting canrenone or a related compound)

In one of the preferred schemes for the preparation of compounds of formula I, the canrenone or a related compound according to formula XIII is used as the starting compound (or, alternatively, this method involves the use of androstenedione or a related compound as the starting compound)

Where:

-A-A- represents the group —CHR ⁴ —CHR ⁵ - or —CR ⁴ = CR ⁵ -;

R ³ , R ⁴ and R ^{5 are} independently selected from the group consisting of hydrogen, halogen, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy;

-B-B- represents a group -CHR ⁶ -CHR ⁷ - or an alpha or beta oriented group:

where R ⁶ and R ^{7 are} independently selected from the group consisting of hydrogen, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; and

R ⁸ and R ^{9 are} independently selected from the group consisting of hydrogen, hydroxy, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy; either R ⁸ and R ⁹ taken together represent a keto, carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ taken together with R ⁶ or R ⁷ represent a carbocyclic or heterocyclic ring structure fused to the pentacyclic ring D.

Using the biological conversion method of the type of the method illustrated in FIGS. 1 and 2, the 11-hydroxy group in the α orientation is introduced into the compound of formula XIII, whereby a compound of formula VIII:

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII.

The compound of formula XIIIA preferably has the structure:

and the 11α-hydroxy product has the structure

in each of which:

-A-A- represents the group —CH ₂ —CH ₂ - or —CH = CH—;

-B-B- represents a group -CH ₂ -CH ₂ - or an α- or β-oriented group:

R ³ represents hydrogen, lower alkyl or lower alkoxy;

X represents two hydrogen atoms, oxo or = S;

Y ¹ and Y ² taken together represent an oxygen bridge —O— or

Y ¹ represents hydroxy, and

Y ² represents hydroxy, lower alkoxy, or, if X represents H ₂ , then lower alkanoyloxy;

and salts of compounds in which X is oxo, and Y ² is hydroxy. More preferably, if the compound of formula VIIIA obtained in this reaction corresponds to formula VIIIA, where each of —A — A—, —B — B— is —CH ₂ —CH ₂ -; R ³ represents hydrogen; Y ¹ and Y ² and X are defined in formula XIIIA; and R ⁸ and R ⁹ taken together form a 20-spiroxane structure:

.

.

Preferred microorganisms that can be used in the indicated hydroxylation step are Aspergillus ochraceus NRRL 405, Aspergillus ochraceus ATCC 18500, Aspergillus niger ATCC 16888 and ATCC 26693, Aspergillus nidulans ATCC 11267, Rhizopus oryzae ATCC11227, RCC, BTCC1122, ATCC11227, RCC, BTCC1122, ATCC11227 , Bacillus megaterium ATCC 14945, Pseudomonas cruciviae ATCC 13262, and Trichothecium roseum ATCC 12543. Other preferred microorganisms are Fusarium oxysporum f.sp.cepae ATCC 11171 and Rhizopus arrhizus ATCC 11145.

Other microorganisms that are active for this reaction are Absidia coerula ATCC 6647, Absidia glauca ATCC 22752, Actinomucor elegans ATCC 6476, Aspergillus flavipes ATCC 1030, Aspergillus fumigatus ATCC 26934, Beauveria bassiana ATCC 7159 and ATaria Tocaeria 14004, Atria, Botria decora ATCC 14767, Chaetomium cochliodes ATCC 10195, Corynespora cassiicola ATCC 16718, Cunninghamella blakesleeana ATCC 8688a, Cunninqhainella echinuata ATCC 3655, Cunninghamella elegans ATCC 9245, Curvularia clavata ATcloc ATCL9 ATCC901 12 butleri ATCC 22822, Hypomyces chrysospermus ATCC IMI 109891, Mortierella isabellina ATCC 42613, Mucor mucedo ATCC 4605, Mucor griseo-cyanus ATCC 1207A, Myrothecium verrucaria ATCC 9095, Nocardia corallina ATCCillomycema Penis 19070, Pacci 19059 patulum ATCC 24550, Pithomyces atro-olivaceus IFO 6651, Pithomyces cynodontis ATCC 26150, Pycnosporium sp. ATCC 12231; Saccharopolyspora erythrae ATCC 11635; Sepedonium chrysospermum ATCC 13378; Stachylidium bicolor ATCC 12672; Streptomyces hygroscopicus ATCC 27438; Streptomyces purpurascens ATCC

Other organisms that are expected to have activity for 11α-hydroxylation are Cephalosporium aphidicola (Phytochemistry (1996), 42 (2), 411-415), Cochliobolus lunatas (J. Biotechnol. (1995), 42 (2) , 145-150), Tieghemella orchidis (Khim-Farm.Zh. (1986), 20 (7), 871-876), Tieghemella hyalospora (Khim.-Farm.Zh. (1986), 20 (7), 871- 876), Monosporium olivaceum (Acta Microbiol. Pol., Ser.B. (1973), 5 (2), 103-110), Aspergillus ustus (Acta Microbiol. Pol., Ser.B. (1973), 5 (2 ), 103-110), Fusarium graminearum (Acta Microbiol.Pol., Ser.B. (1973), 5 (2), 103-110), Verticillium glaucum (Acta Microbiol.Pol., Ser.B (1973), 5 (2), 103-110) and Rhizopus nigricans (J. Steroid Biochem. (1987), 28 (2), 197-201).

11β-Hydroxy derivatives of androstenedione and mexrenone can be obtained by the biological conversion methods described in Examples 19A and 19B, respectively. By analogy, the authors of the present invention have suggested that the corresponding β-hydroxy isomer of a compound of formula VIII having, instead of a C11-α-hydroxy substituent, a C11-β-hydroxy substituent can be obtained by a similar biological conversion method using suitable microorganisms, capable of 11β-hydroxylation, such as one or more of the microorganisms described in this application.

Before carrying out large-scale fermentation for the hydroxylation of carnenone or other substrates of the formula XIII, a cell inoculum is obtained in a seed fermentation system containing a seed fermenter, or two or more series-connected seed fermenters. A working stock spore suspension is introduced into the first seed fermenter along with a nutrient solution for cell culture. If it is desired or necessary that the volume of the inoculum exceeds the volume that is produced in the first seed fermenter, then this volume of the inoculum can be increased in arithmetic or geometric progression by passing through the remaining sequentially located fermenters in the seed fermentation unit. Preferably, the inoculum produced in the seed system for fermentation has a sufficient volume and contains viable cells to achieve rapid initiation of the reaction in the production fermenter; and that consecutive production cycles are relatively short, and also that the fermenter has high productivity. Regardless of the number of vessels in the seed fermenter system, the second and subsequent seed fermenters should preferably be of such a size that the degree of dilution in each stage of this series is essentially the same. The initial dilution of the inoculum in each seed fermenter may be approximately the same as the dilution in the production fermenter. Canrenone or another substrate of formula XIII is loaded into the production fermenter together with the inoculum and nutrient solution and a hydroxylation reaction is carried out.

A spore suspension loaded into the seed system for fermentation is supplied from a vessel with a working stock solution of a spore suspension taken from a plurality of vessels containing an already prepared working stock of cells, which, before use, is stored under cryogenic conditions. This working stock of source cells, in turn, comes from the uterine stock of source cells, which is obtained as follows. A spore sample obtained from an appropriate source, for example from ATCC, is first suspended in an aqueous medium, such as, for example, saline, nutrient solution or a solution of a surfactant (e.g., a nonionic surfactant, such as Tween 20, at a concentration about 0.001% by weight) and this suspension is distributed into culture dishes, each containing a solid nutrient mixture, usually based on a non-hydrolyzable polysaccharide, such as agar, where spores propagate. The solid nutrient mixture preferably contains from about 0.5% to about 5% of the mass. glucose, from about 0.05% to about 5% of the mass. a nitrogen source, for example peptone; from about 0.05% to about 0.5% of the mass. a source of phosphorus, for example ammonium phosphate or an alkali metal such as dipotassium biphosphate; from about 0.25% to about 2.5% of the mass. yeast lysate or extract (or other source of amino acid, such as meat extract or broth with cardiac extract); from about 1% to about 2% of the mass. agar or another non-hydrolyzable polysaccharide. In addition, but not necessarily, the solid nutrient mixture may contain and / or contains from about 0.1% to about 5% of the mass. malt extract. The pH of this solid nutrient mixture is preferably from about 5.0 to about 7.0, and if necessary, it can be adjusted by adding alkali metal hydroxide or phosphoric acid. Suitable solid culture media are the following:

1. Solid Medium # 1 1% glucose, 0.25% yeast extract, 0.3% K ₂ HPO ₄ and 2% agar (Bacto); pH adjusted to 6.5 with 20% NaOH 2. Solid medium # 2 2% peptone (Bacto), 1% yeast extract (Bacto), 2% glucose and 2% agar (Bacto); pH adjusted to 5 with 10% H ₃ PO ₄ 3. Solid medium # 3 0.1% peptone (Bacto), 2% malt extract (Bacto), 2% glucose and 2% agar (Bacto); pH = 5.3 4. Liquid medium 5% black molasses, 0.5% liquid corn extract, 0.25% glucose, 0.25% NaCl and 0.5% KH ₂ PO ₄ , pH = 5.8 5. Mycological agar Difco (low pH)

The number of agar plates used to produce the main starting cell stock can be selected depending on future requirements for the starting stock material, but generally this number is from about 15 to about 30 cups thus prepared. After an appropriate cultivation period, for example for 7-10 days, the plates are scrubbed to collect spores in the presence of an aqueous carrier, usually physiological saline or buffer, and the resulting uterine stock suspension is distributed into small vessels, for example, 1 ml of the suspension is placed in each of the sets 1, 5 ml of blood vessels. In order to obtain a working initial spore suspension for research or for industrial fermentation, the contents of one or more of these vessels with second generation mother liquors can be distributed into agar plates and incubated in a manner analogous to the method described above for obtaining the mother liquor spore stock. If traditional industrial production is contemplated, then at least 100 to 400 cups may be used to produce a second generation working feed. The contents of each cup are scrubbed into a separate vessel with the working starting material, where each vessel usually contains 1 ml of the produced inoculum. For constant preservation, both the uterine initial suspension and the second generation inoculum are stored mainly in the vapor phase in a cryogenic storage vessel containing liquid N ₂ or other cryogenic liquid.

In the method illustrated in FIG. 1, an aqueous culture medium is obtained that contains a nitrogen source, such as peptone, a yeast derivative or its equivalent, glucose, and a phosphorus source, such as a phosphate salt. The spores of the microorganism are cultured in this medium in a seed fermentation system. A preferred microorganism is Aspergillus ochraceus NRRL 405 (ATCC 18500). Then, the seed medium thus obtained is introduced into the fermenter for production together with the substrate of the formula XIII. The fermentation broth is stirred and aerated for an interval of time sufficient to complete the reaction to the desired degree of completion.

The medium for the seed fermenter preferably includes an aqueous mixture that contains from about 0.5% and about 5% of the mass. glucose, from about 0.05% to about 5% of the mass. a nitrogen source, for example peptone; from about 0.05% to about 0.5% of the mass. a source of phosphorus, for example ammonium phosphate or an alkali metal such as monobasic ammonium phosphate or dipotassium bisphosphate; from about 0.25% to about 2.5% of the mass. a yeast lysate or extract (or other source of an amino acid, such as bard extract); from about 1% to about 2% of the mass. agar or another non-hydrolyzable polysaccharide. A particularly preferred medium for cultivation of seed contains from about 0.05% to about 5% of the mass. a nitrogen source such as peptone; from about 0.25% to about 2.5% of the mass. autolized yeast or yeast extract; from about 0.5% to about 5% of the mass. glucose and from about 0.05% to about 0.5% of the mass. a source of phosphorus such as monobasic ammonium phosphate. Particularly economical methods have been developed using another preferred seed crop, which contains from about 0.5% to about 5% of the mass. liquid corn extract, from about 0.25% to about 2.5% of the mass. autolized yeast or yeast extract; from about 0.5% to about 5% of the mass. glucose and from about 0.05% to about 0.5% of the mass. monobasic ammonium phosphate. Liquid corn extract is a particularly economical source of proteins, peptides, carbohydrates, organic acids, vitamins, metal ions, trace elements and phosphates. Instead of liquid corn extract or in addition to it, a solution of pulp from other grains can be used. The pH of this medium is preferably adjusted to from about 5.0 to about 7.0, for example by adding alkali metal hydroxide or phosphoric acid. If a liquid corn extract is used as a source of nitrogen and carbon, then the pH is preferably adjusted to a value from about 6.2 to about 6.8. The medium containing peptone and glucose is preferably adjusted to a pH of from about 5.4 to about 6.2. Cultivation media suitable for use in seed fermentation are:

1. Wednesday # 1 2% peptone, 2% autolysed yeast (or yeast extract) and 2% glucose; pH adjusted to 5.8 using 20% NaOH 2. Wednesday # 2 3% liquid corn extract, 1.5% yeast extract, 0.3% monobasic ammonium phosphate and 3% glucose, pH adjusted to 6.5 using 20% NaOH.

The spores of the microorganism are introduced into this medium from a vessel, usually containing approximately 10 ⁹ spores per ml of suspension. The optimal productivity of seed culture is achieved when, when diluted with the culture medium at the beginning of the cultivation of the seed culture, the spore population density does not decrease below about 10 ⁷ per ml. Preferably, the spores are cultured in a seed fermentation system until the volume of precipitated mycelium (PMV) in the seed fermenter is at least about 20%, and preferably from about 35% to about 45%. Since the cycle in a seeding fermentation vessel (or in any vessel of the plurality of vessels that make up the seed fermentation system) depends on the initial concentration in that vessel, it may be preferable to carry out two or three stages of seed fermentation to speed up the whole process. However, it is advisable to avoid the use of significantly more than three fermenters in the system, since when the fermentation of the seed involves an excessive number of stages, this can adversely affect the activity of the process. The fermentation of the seed culture is carried out with stirring at a temperature in the range from about 23 to about 37 ° C, and preferably in the range from about 24 to about 28 ° C.

The culture from the seed fermentation system is introduced into the production fermenter along with the production culture medium. In one embodiment, the non-sterile canrenone or other substrate of formula XIII is used as a substrate for the reaction. Preferably, if the substrate is added to the production fermenter in the form of 10% -30% of the mass. suspensions in the medium for cultivation. To increase the surface area available for the 11α-hydroxylation reaction, before the introduction of the substrate of the formula XIII into the fermenter, the particle size of this substrate is reduced by passing it through a stand-alone micronizer (fine grinding mill). A sterile stock nutrient solution containing glucose and a second sterile nutrient solution containing a yeast derivative such as autolysed yeast (or an equivalent amino acid composition based on alternative sources such as bard extract) are also separately administered. This medium includes an aqueous mixture containing from about 0.5% and about 5% of the mass. glucose, from about 0.05% to about 5% of the mass. a nitrogen source, for example peptone; from about 0.05% to about 0.5% of the mass. a source of phosphorus, for example ammonium phosphate, or an alkali metal such as dipotassium biphosphate; from about 0.25% to about 2.5% of the mass. a yeast lysate or extract (or other source of an amino acid, such as bard extract); from about 1% to about 2% of the mass. agar or another non-hydrolyzable polysaccharide. A particularly preferred production medium for cultivation contains from about 0.05% to about 5% of the mass. a nitrogen source, for example peptone; from about 0.25% to about 2.5% of the mass. autolized yeast extract; from about 0.5% to about 5% of the mass. glucose and from about 0.05% to about 0.5% of the mass. a source of phosphorus such as monobasic ammonium phosphate. Another preferred production environment contains from about 0.5% to about 5% of the mass. liquid corn extract, from about 0.25% to about 2.5% of the mass. autolized yeast or yeast extract; from about 0.5% to about 5% of the mass. glucose and from about 0.05% to about 0.5% of the mass. monobasic ammonium phosphate. The pH of the medium for production fermentation is preferably adjusted by the method described above for the medium intended for inoculum fermentation, the pH ranges indicated for the medium containing peptone / glucose and the medium containing liquid corn extract, respectively, being the most preferred. Cultivation media suitable for biological conversion reactions are indicated below:

1. Wednesday # 1 2% peptone, 2% autolysed yeast (or yeast extract) and 2% glucose; pH adjusted to 5.8 with 20% NaOH 2. Wednesday # 2 1% peptone, 1% autolysed yeast (or yeast extract) and 2% glucose; pH adjusted to 5.8 with 20% NaOH 3. Wednesday # 3 0.5% peptone, 0.5% autolysed yeast (or yeast extract) and 0.5% glucose, pH adjusted to 5.8 using 20% NaOH. 4. Wednesday # 4 3% liquid corn extract, 1.5% yeast extract, 0.3% monobasic ammonium phosphate and 3% glucose, pH adjusted to 6.5 with 20% NaOH. 5. Wednesday # 5 2.55% liquid corn extract, 1.275% yeast extract, 0.255% monobasic ammonium phosphate and 3% glucose, pH adjusted to 6.5 with 20% NaOH. 6. Wednesday # 6 2.1% liquid corn extract, 1.05% yeast extract, 0.21% monobasic ammonium phosphate and 3% glucose, pH adjusted to 6.5 with 20% NaOH.

The non-sterile canrenone solution and the sterile nutrient solution are charged by chain feeding into the production fermenter from about five to about twenty, preferably from about ten to about fifteen servings, and preferably substantially equal portions of each ingredient throughout the production cycle. Moreover, before inoculation with broth for seed fermentation, the substrate is first introduced, preferably in an amount sufficient to achieve a concentration of from about 0.1% of the mass. up to about 3% of the mass., and preferably from about 0.5% of the mass. up to about 2% wt., and then periodically added, mainly every 8-24 hours, to achieve a cumulative amount of from about 1% to about 8% of the mass. If an additional amount of the substrate is added every 8 hours, then the total addition will be slightly less, for example 0.25% -2.5% by weight, than in the case when the substrate is added only after a day. In the latter case, it may be necessary cumulative addition of canrenone in the range from 2% to about 8% of the mass. An additional nutrient mixture added during the fermentation reaction is preferably a concentrate, for example a mixture containing from about 40% to about 60% of the mass. sterile glucose, from about 16% to about 32% of the mass. a sterile yeast extract or other sterile source of a yeast derivative (or other amino acid source). Since the substrate fed to the production fermenter shown in FIG. 1 is non-sterile, antibiotics are periodically added to the fermentation broth to inhibit the growth of undesirable microorganisms. Antibiotics such as kanamycin, tetracycline and cephalexin can be added without any adverse effect on the growth and biological conversion of microorganisms. These antibiotics are added to the fermentation broth, preferably at a concentration of, for example, from about 0.0004% to about 0.002% of the total weight of the broth, containing, for example, from about 0.0002% to about 0.0006% kanamycin sulfate, from about 0.0002% to about 0.006% tetracycline · HCl and / or from about 0.001% to about 0.003% cephalexin based on the total weight of the broth.

Typically, a production fermentation cycle lasts approximately 80-160 hours. Thus, portions of each of the substrates of formula XIII and nutrient solutions are usually added approximately every 2-10 hours, and preferably every 4-6 hours. It is also preferable to introduce an antifoam agent in the seed fermentation system and in the production fermenter.

In the method illustrated in FIG. 1, the inoculum is loaded into a production fermenter, preferably in an amount of from about 0.5% to about 7%, more preferably from about 1% to about 2%, by volume of the total mixture in the fermenter, and the glucose concentration is maintained from about 0.01% to about 1.0%, preferably from about 0.025% to about 0.5%, and more preferably from about 0.05% to about 0.25% by weight, by periodic additions, which are preferably carried out in portions from about 0.05% to about 0.25% by weight of the full batch of the load. The fermentation temperature is preferably controlled in the range from 20 ° C to 37 ° C, preferably from 24 ° C to about 28 ° C, however, it may be desirable to gradually lower the temperature during the reaction, for example by 2 ° C, but at the same time maintain the volume of precipitated mycelium (PMV) below about 60%, and more preferably below about 50%, which helps to prevent the increase in viscosity of the broth during fermentation, which may prevent sufficient stirring. If the growth of biomass extends beyond the surface of the liquid, then the substrate present in the biomass may be outside the reaction zone and may not be available for the hydroxylation reaction. To maintain productivity, it is desirable that the PMV reaches 30-50%, and preferably 35-45% in the first 24 hours of the fermentation reaction, and then the conditions are preferably adjusted to regulate subsequent growth within the limits set above. During the reaction, the pH of the fermentation medium is adjusted within the range of from about 5.0 to about 6.5, preferably from about 5.2 to about 5.8, and the contents of the fermenter are stirred at a speed of from about 400 to about 800 rpm. The level of dissolved oxygen at least about 10% of saturation is achieved by aeration of the batch at about 0.2-1.0 rpm / min, while the pressure in the head of the fermenter is approximately in the range from atmospheric pressure to about 1.0 bar and more preferably of the order of about 0.7 bar. The stirring speed can also be increased if necessary to maintain minimum levels of dissolved oxygen. Dissolved oxygen is maintained mainly at a level significantly exceeding about 10%, and in fact up to about 50% to stimulate the conversion of the substrate. Maintaining a pH within 5.5 ± 0.2 is also an optimal condition for biological conversion. Foaming is controlled, if necessary, by adding a commonly used antifoam. After adding the entire substrate, the reaction is preferably continued until the molar ratio of the product of formula VIII to the remaining unreacted substrate of formula XIII is at least about 9 to 1. This conversion can be achieved during the 80-160-hour cycle described higher.

It was found that a high degree of conversion is associated with the depletion of the levels of the original nutrients below the initial level of loading, and therefore, by regulating the speed of aeration and the speed of mixing, it is possible to prevent spilling of the substrate from the liquid broth. In the process illustrated in FIG. 1, the nutrient level is depleted and then maintained at a level not exceeding about 60%, preferably about 50%, of the initial load, and in the processes illustrated in FIGS. 2 and 3, the nutrient level substances is reduced to a level and maintained at a level not exceeding about 80%, preferably about 70% of the initial load. The aeration speed is preferably not more than 1 rpm / min, and more preferably about 0.5 rpm / min; and the stirring speed is preferably not more than 600 rpm.

A particularly preferred process for preparing a compound of formula VIII is illustrated in FIG. 2. A preferred microorganism for 11α-hydroxylation of a compound of formula XIII (e.g., canrenone) is Aspergillus ochraceus NRRL 405 (ATCC 18500). In this method, the culture medium preferably contains from about 0.5% to about 5% of the mass. liquid corn extract, from about 0.5% to about 5% of the mass. glucose, from about 0.1% to about 3% of the mass. yeast extract and from about 0.05% to about 0.5% of the mass. ammonium phosphate. However, other culture media may also be used. Inoculum is obtained mainly by the method illustrated in FIG. 1, using any seed fermentation medium described herein. A suspension of unmilled canrenone or another substrate of Formula XIII in a culture medium is prepared under aseptic conditions in a mixer, preferably at a relatively high concentration of about 10% to about 30% by weight of the substrate. Preferably, aseptic preparation conditions may include sterilization or pasteurization of the suspension after mixing. The full amount of a sterile suspension of the substrate required to obtain a batch of product is introduced into the production fermenter at the beginning of the cycle or by periodic chain feeding. The particle size of the substrate is reduced by wet grinding in a pump with a shear action, operating in an operational mode, which feeds the suspension into the production fermenter, which avoids the need to use a stand-alone micronizer. If aseptic conditions are ensured by pasteurization rather than sterilization, the degree of agglomeration may be insignificant, however, the use of a shear pump may be desirable for positive control of particle size. The sterile culture medium and the glucose solution are introduced into the production fermenter, mainly by a method similar to that described above. All nutrient components are sterilized before being introduced into the production fermenter, and therefore antibiotic use is not required.

In a preferred embodiment of the method illustrated in FIG. 2, the inoculum is introduced into the production fermenter in an amount of from about 0.5% to about 7%, wherein the fermentation temperature is from about 20 ° C. to about 37 ° C., preferably from about 24 ° C to about 28 ° C, and the pH is adjusted in the range from about 4.4 to about 6.5, and preferably from about 5.3 to about 5.5, for example, by introducing gaseous ammonia, aqueous ammonium hydroxide, aqueous alkaline hydroxide metal or phosphoric acid. As in the method shown in FIG. 1, the temperature is preferably adjusted to control biomass growth so that the PMV does not exceed 55-60%. The initial loading of glucose is preferably from about 1% to about 4% by weight, and more preferably 2.5% -3.5% by weight, however, during the fermentation process, it can preferably be below about 1.0% by weight. An additional amount of glucose is periodically supplied in portions from about 0.2% to about 1.0% based on the total weight of the batch so that the glucose concentration in the fermentation zone is maintained in the range from about 0.1% to about 1.5% by weight, and preferably from about 0.25% to about 0.5% of the mass. Sources of nitrogen and phosphorus may, but are not necessarily, be supplied with glucose. However, since the loading of all canrenone is performed at the beginning of the cycle for a given patria, the necessary supply of nitrogen and phosphorus-containing nutrients can also be carried out simultaneously, which will allow using only a glucose solution for addition during the reaction. The speed and type of stirring varies greatly. Moderate stirring stimulates mass transfer between the solid substrate and the aqueous phase. However, to prevent decomposition of the myelin of microorganisms, it is necessary to use a stirrer with small shear forces. The optimal stirring speed varies from 200 to 800 rpm depending on the viscosity of the culture broth, oxygen concentration and mixing conditions, which are influenced by the configuration of the vessel, septum and mixer. Generally, a preferred stirring speed is in the range of 350-600 rpm. Preferably, the stirring blades perform the function of axial pumping from top to bottom, which ensures good mixing of the fermented biomass. This batch is preferably aerated at a speed of from about 0.3 to about 1.0 rpm / min, and preferably from about 0.4 to 0.8 about rpm / min, and the pressure in the head of the fermenter is preferably from about 0.5 to about 1.0 bar on a reference scale. The stirring temperature, aeration and back pressure are preferably controlled to maintain the amount of dissolved oxygen at least about 10% by volume during the biological conversion. The duration of the entire cycle for a given batch is usually from about 100 to about 140 hours.

Although the principle of the method illustrated in FIG. 2 is based on the early introduction of basically the entire canrenone load, however, it should be noted that cultivation of the fermentation broth can be carried out before loading the entire canrenone volume. Some of the canrenone may also be added to the batch later, but not necessarily. However, generally, 48 hours after the initiation of fermentation, at least about 75% sterile canrenone should be introduced into the fermenter for transformation. Moreover, it is desirable that at least about 25% of the mass. canrenone was introduced at the beginning of fermentation or at least in the first 24 hours of fermentation to stimulate the production of the biological conversion enzyme (s).

In a more preferred method, illustrated in FIG. 3, the full batch and nutrient solution are sterilized in a production fermentation apparatus, and then the inoculum is introduced. Nutrient solutions that can be used, as well as preferred of these solutions, are basically the same as in the method illustrated in FIG. 2. In this embodiment, the shear force of the agitator blades destroys the agglomerates of the substrate, which in one way or another form after sterilization. It was found that this reaction proceeds favorably if the particle size of canrenone is less than about 300 microns and at least 75% of the mass. all particles have a size of less than 240 microns. It was found that the use of a suitable mixer, for example a disk turbine mixer, with an adequate speed of about 200-800 rpm, with a maximum speed of at least about 400 cm / sec, provides a shear rate sufficient to maintain the indicated particle size values, despite the agglomeration of particles that usually occurs after sterilization in a production fermenter. The remaining operations of the method illustrated in FIG. 3 are basically the same as in the method illustrated in FIG. 2. The methods illustrated in FIGS. 2 and 3 have some significant advantages over the method illustrated in FIG. 1. The main advantage is the ability to use an inexpensive nutritional base such as liquid corn extract. Other advantages are realized by eliminating the need to add antibiotics, which simplifies the feeding procedures and allows sterilization of a batch of canrenone or another substrate of the formula XIII. Another significant advantage is the ability to use a simple glucose solution instead of a complex nutrient solution to add during the reaction cycle.

In the methods illustrated in FIGS. 1-3, the product of formula VIII is a crystalline solid that, together with biomass, can be isolated from the reaction broth by filtration or low-speed centrifugation. Alternatively, this product can be extracted from the entire reaction broth using organic solvents. The product of formula VIII is isolated by solvent extraction. To maximize recovery, the liquid phase filtrate and the biomass filter or centrifuge sediment are treated with an extracting solvent, but usually ≥95% of the product is associated with biomass. Typically, hydrocarbons, esters, chlorinated hydrocarbons and ketone solvents may be used for extraction. A preferred solvent is ethyl acetate. Other mainly suitable solvents are toluene and methyl isobutyl ketone. For extraction from the liquid phase, it may be preferable to use a volume of solvent approximately equal to the volume of the reaction solution with which it is in contact. To recover the product from biomass, this biomass is suspended in a solvent, preferably in a large excess relative to the initial loading of the substrate, for example 50-100 ml of solvent per gram of the initial loading of canrenone, and the resulting suspension is preferably heated under reflux for a period of about 20 minutes to several hours to guarantee the transfer of the product to the solvent phase from the recesses and pores of the biomass. After that, the biomass is removed by filtration or centrifugation, and the filter cake is preferably washed with fresh solvent and deionized water. Then the aqueous washing and washing with a solvent are combined and left to separate phases. The product of formula VIII is isolated by crystallization from solution. To maximize yield, the mycelium is contacted twice with a fresh solvent. After precipitation, the product is isolated from the solvent phase to completely separate the aqueous phase. More preferably, the solvent is evaporated under vacuum until crystallization begins, and then the concentrated extract is cooled to a temperature of from about 0 ° C to about 20 ° C, and preferably from about 10 ° C to about 15 ° C, over a period time sufficient for precipitation and crystal growth, usually within about 8 to about 12 hours.

Most preferred are the methods illustrated in FIG. 2, and especially the method illustrated in FIG. 3. These methods are carried out at low viscosity and are suitable for the precise control of process parameters, such as pH, temperature and level of dissolved oxygen. In addition, sterility conditions are easily maintained without antibiotics.

Biological conversion processes are exothermic, which requires heat removal using a jacketed fermenter or a cooling coil in a production fermenter. Alternatively, the reaction broth may be recycled through an external heat exchanger. The dissolved oxygen is preferably maintained at least about 5%, and preferably at least about 10% by volume, which is sufficient to provide energy for the reaction and to guarantee the conversion of glucose to CO ₂ and H ₂ O by controlling the flow rate air introduced into the reactor after measuring the oxygen level in the broth. The pH is preferably maintained in the range of about 4.5 to about 6.5.

In each of the alternative methods for 11-hydroxylating a substrate of formula XIII, the productivity of this method is limited by mass transfer from the solid substrate to the aqueous phase or to the phase boundary region where the reaction is obviously taking place. As indicated above, productivity is generally not limited by the mass transfer rate, provided that the average particle size of the substrate is reduced to less than about 300 microns, and that at least 75% of the particles are smaller than 240 microns. However, the productivity of these processes can be further enhanced in some alternative embodiments of the invention in which the main loading of canrenone or another substrate of formula XIII is provided in a production fermenter in an organic solvent. According to this embodiment, the substrate is dissolved in a water-immiscible solvent and mixed with the starting aqueous culture medium for reseeding and with the surfactant. Suitable water-immiscible solvents are, for example, DMF, DMSO, C ₆ -C ₁₂ fatty acids, C ₆ -C ₁₂ alkanes, vegetable oils, sorbitans and aqueous surfactant solutions. Stirring of this charge contributes to the production of an emulsion reaction system having an extended interfacial area for transferring the mass of the substrate from the organic liquid phase to the reaction zones.

In a second embodiment, the substrate is first dissolved in a water-miscible solvent, such as acetone, methyl ethyl ketone, methanol, ethanol or glycerol, at a concentration substantially exceeding its solubility in water. Upon receipt of the initial substrate solution at an elevated temperature, the solubility increases, which contributes to an increase in the amount of the soluble form of the substrate introduced into the reactor, and thereby increases the payload of the reactor. A warm substrate solution is charged to a production fermentation reactor together with a relatively cold aqueous charge containing culture medium and inoculum. When mixing the substrate solution with the aqueous medium, the substrate precipitates. However, under conditions of significant super-saturation and moderately intense stirring, instead of crystal growth, the predominant formation of crystallization centers occurs, and very small particles with a large surface area are formed. A large surface area stimulates mass transfer between the liquid phase and the solid substrate. In addition, the equilibrium concentration of the substrate in the aqueous liquid phase also increases in the presence of a water-miscible solvent. Accordingly, productivity increases.

Although this microorganism does not have to be tolerant to a high concentration of organic solvent in the aqueous phase, it is desirable to use a concentration of ethanol, for example, in the range of from about 3% to about 5% by weight.

A third embodiment of the invention is the solubilization of the substrate in an aqueous solution of cyclodextrin. Typical cyclodextrins are hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratio of substrate: cyclodextrin may be from about 1: 0.5 to about 1: 1.5, and more preferably from about 1: 0.8 to about 1: 1. Then this substrate: cyclodextrin mixture can be added, under aseptic conditions, to a reactor for biological conversion.

11α-Hydroxycanrenone and other 11α-hydroxylation reaction products (Formulas VIII and VIIIA) are new compounds that can be isolated by filtering the reaction medium and extracting the product from biomass collected on the medium after filtration. For extraction, standard organic solvents can be used, for example ethyl acetate, acetone, toluene, chlorinated hydrocarbons and methyl isobutyl ketone. The product of formula VIII can then be recrystallized from a similar type of organic solvent. The compounds of formula VIII are mainly used as intermediates for the preparation of compounds of formula I and, mainly, compounds of formula IA.

Preferably, the compounds of formula VIII correspond to formula VIIIA, where -A-A- and -B-B- are -CH ₂ -CH ₂ -, R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20 Spiroxane ring:

Then, in accordance with the method shown in Scheme 1, the compound of formula VIII is reacted with a source of cyanide ion under alkaline conditions to obtain an enamine compound of formula VII

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above.

If the substrate corresponds to formula VIIIA, then the product is a compound of formula VIIA:

where -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA.

R ³ is preferably hydrogen.

The cyanidation of the 11α-hydroxyl substrate of formula VIII can be carried out by reacting it with a source of cyanide ion, such as ketone cyanohydrin, most preferably acetone cyanohydrin, in the presence of a base and an alkali metal salt, most preferably LiCl.

Alternatively, cyanidation can be carried out without cyanohydrin using alkali metal cyanide in the presence of acid.

In the method using keto cyanohydrin, the reaction is carried out in solution, preferably using an aprotic polar solvent such as dimethylformamide or dimethyl sulfoxide. The formation of enamine requires the use of at least two moles of the source of cyanide ion per mole of substrate, and preferably a small excess of the source of cyanide. The base is preferably a nitrogenous base such as dialkylamine, trialkylamine, alkanolamine, pyridine or the like. However, inorganic bases such as alkali metal carbonates or alkali metal hydroxides may also be used. Preferably, the substrate of formula VIII is initially present in a ratio of from about 20 to about 50% by weight, and the base is present in a ratio of from 0.5 to two equivalents per equivalent of substrate. The reaction temperature does not play a decisive role, but at elevated temperature the yield of the product increases. So, for example, if triethylamine is used as the base, the reaction is preferably carried out in the temperature range from about 80 ° C to about 90 ° C. At this temperature, the reaction proceeds to its completion in a period of from about 5 to about 20 hours. If diisopropylethylamine is used as the base, the reaction is carried out at 105 ° C, and this reaction is completed after 8 hours. At the end of the reaction, the solvent is removed in vacuo and the residual oil is dissolved in water and neutralized to pH 7 by the addition of dilute acid, preferably hydrochloric acid. The product precipitates from this solution, after which it is washed with distilled water and air dried. The release of HCN can be stopped by inert gas purging and quenched in an alkaline solution. The dried precipitate is dissolved in chloroform or another suitable solvent, and then extracted with concentrated acid, for example 6N HCl. The extract is neutralized to pH 7 by adding an inorganic base, preferably an alkali metal hydroxide, and cooled to a temperature within 0 ° C. The precipitate obtained is washed and dried, and then recrystallized from a suitable solvent, for example acetone, to give a product of formula VII, which can be used in the next step.

Alternatively, the reaction can be carried out in an aqueous solvent system containing a water-miscible organic solvent, such as methanol, or in a two-phase system containing water and an organic solvent, such as ethyl acetate. In this alternative embodiment, the product can be isolated by diluting the reaction solution with water, followed by extraction of the product using an organic solvent such as methylene chloride or chloroform, and then back extraction from the organic extract using concentrated mineral acid, for example 2N HCl. See US Patent No. 3200113.

According to another alternative method, the reaction can be carried out in a water-miscible solvent, such as dimethylformamide, dimethylacetamide, N-methyl, pyrrolidone or dimethyl sulfoxide, after which the solution of the reaction product is diluted with water and made alkaline, for example by adding alkali metal carbonate, and then cooled to 0-10 ° C, which leads to precipitation of the product. Preferably, the system is quenched with alkali metal hypogalite or another reagent effective to prevent cyanide release. After filtration and washing with water, the precipitated product can be used in a subsequent step of the process.

According to yet another alternative method, an enamine product of formula VII can be prepared by reacting a substrate of formula VIII, in the presence of a proton source, with an excess of alkali metal cyanide, preferably NaCN, in an aqueous solvent containing an aprotic, water-miscible polar solvent such as dimethylformamide or dimethylacetamide. The proton source is preferably a mineral acid or a C ₁ -C ₅ carboxylic acid, and sulfuric acid is most preferred. It is interesting to note that if the cyanidating reagent is the commercially available LiCN reagent in DMF, then a discrete proton source is not necessary.

A source of cyanide ions, such as an alkali metal salt, is charged to the reactor, preferably in a ratio of from about 2.05 to about 5 molar equivalents per equivalent of substrate. It is obvious that mineral acid, or another source of protons, stimulates the attachment of HCN at 4,5- and 6,7-double bonds and is preferably present in the ratio of at least one molar equivalent per molar equivalent of the substrate; but the reaction system must remain alkaline, by maintaining an excess of alkali metal cyanide in relation to the amount of acid present. The reaction is preferably carried out at a temperature of at least about 75 ° C, usually at 60 ° C-100 ° C, for a period of time from about 1 hour to about 8 hours, and preferably from about 1.5 to about 3 hours. At the end of the reaction, the reaction mixture is cooled, preferably to about room temperature; and the enamine product is precipitated by acidifying the reaction mixture and mixing it with cold water, preferably at a temperature close to the temperature of an ice bath. Acidification obviously favors the closure of 17-lactone, which tends to open under the alkaline conditions prevailing in the cyanidation reaction. The reaction mixture is usually acidified using the same acid that is present in the reaction and which is preferably sulfuric acid. Water is preferably added in a ratio of from about 10 to about 50 molar equivalents per mole of product.

The compounds of formula VII are new compounds and are used mainly as intermediates for the preparation of compounds of formula I, and especially of formula IA. Preferably, the compounds of formula VII correspond to formula VIIA, where -A-A- and -B-B- are -CH ₂ -CH ₂ -, R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20 Spiroxane ring:

The most preferred compound of formula VII is 5'R (5'α), 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'α, 13'α-dimethyl-3 ', 5'-dioxospiro [furan- 2 (3H), 17'α (5'H) - [7,4] metheno [4H] cyclopenta [a] phenanthrene] -5'-carbonitrile.

In the reaction of converting a compound of formula VIII to enamine of Formula VII in a crude product, a 7-cyano derivative of a compound of formula VIII was observed by chromatography. It has been suggested that in this conversion process, the 7-cyano compound is an intermediate. In addition, it was hypothesized that the reaction of the 7-cyano derivative itself leads to the formation of a second intermediate compound, a 5,7-dicyano derivative of the formula VIII, which in turn reacts to form an ester. See, for example, R. Christiansen et al., The Reaction of Steroidal 4,6-Dien-3-Ones With Cyanide, Steroids, Vol. 1, June 1963, which is incorporated herein by reference. These new compounds are also used as chromatographic markers, and are also synthetic intermediates. In a preferred embodiment of this complete synthesis step shown in Scheme 1, these intermediates are 7α-cyano-11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-21-dicarboxylic acid, γ-lactone and 5β , 7α-dicyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-21-dicarboxylic acid, γ-lactone.

In the next synthesis step shown in Scheme 1, the enamine of formula VII is hydrolyzed to give a diketone compound of formula VI:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII.

For hydrolysis, any aqueous organic or mineral acid may be used. Hydrochloric acid is preferred. To increase the yield of the product, a water-miscible organic solvent, such as dimethylacetamide or lower alkanol, is preferably used as a co-solvent. A more preferred solvent is dimethylacetamide. Acid must be present in a ratio of at least one equivalent per equivalent of a substrate of formula VII. In the aqueous system, the enamine VII substrate can be substantially converted to the diketone of formula VI for about 5 hours at about 80 ° C. Carrying out the reaction at elevated temperature increases the yield of the product, but the temperature does not play a decisive role. A suitable temperature is selected based on the volatility of the solvent system and the acid.

Preferably, the enamine substrate of formula VII corresponds to formula VIIA:

and the diketone product corresponds to the formula VIA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA. Preferably, R ³ is hydrogen.

At the end of the reaction, the solution is cooled to a temperature of from about 0 ° C to 25 ° C to crystallize the product. The product in the form of crystals can be recrystallized from a suitable solvent, such as isopropanol or methanol, to obtain a product of formula VI, which can be used in the subsequent stage of this method; but usually recrystallization is not a necessary stage. The products of formula VI are new compounds that are mainly used as intermediates for the preparation of compounds of formula I, and especially compounds of formula IA. Preferably, compounds of formula VI correspond to compounds of formula VIA, where —A — A— and —B — B— are —CH ₂ —CH ₂ -, R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20-spiroxane ring:

The most preferred compound of formula VI is 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-trioxospiro [furan-2 (3H ), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile.

In a most preferred embodiment of the present invention, an enamine product of formula VII is prepared from a compound of formula VIII by the method described above and converted in situ into a diketone of formula VI. In this embodiment of the present invention, the substrate of formula VIII is reacted with an excess of alkali metal cyanide in an aqueous solvent containing a proton source, or, optionally, with an excess of ketone cyanohydrin in the presence of a base and LiCI, as described above. However, instead of cooling the reaction mixture, acidification and addition of water are carried out in a ratio necessary for the precipitation of enamine, while significant cooling of the reaction mixture is preferably avoided. Instead, at the end of the cyanidation reaction, water and an acid, preferably a mineral acid such as sulfuric acid, are added to the mixture. This amount of acid added is sufficient to neutralize the excess alkali metal cyanide, which usually requires the introduction of at least one molar equivalent of acid per mol of substrate of formula VIII, and preferably from about 2 to about 5 molar equivalents of acid per equivalent of substrate. However, in order to avoid significant precipitation, the reaction is carried out at a sufficiently high temperature and with a sufficiently large dilution, and the hydrolysis of enamine into diketone is carried out in the liquid phase. Thus, this reaction process proceeds with minimal interruptions and gives a high yield of product. The hydrolysis is preferably carried out at a temperature of at least 80 ° C, more preferably in the range of from about 90 ° C to about 100 ° C, over a period of time, usually from about 1 hour to about 10 hours, and more preferably from about 2 to about 5 hours. Then the reaction mixture is cooled, preferably to a temperature of from about 0 ° C to about 15 ° C, mainly in an ice bath at a temperature of from about 5 ° C to about 10 ° C, to precipitate a diketone product of formula VI. This solid product can be isolated by filtration, and a reduction in the amount of impurities is achieved by washing with water.

In the next step in the synthesis of Scheme 1, the diketone compound of formula VI is reacted with a metal alkoxide to open the ketone bridge between the 4- and 7-positions by breaking the bond between the carbonyl group and the 4-carbon to form an α-oriented alkoxycarbonyl substituent at the 7-position and removing cyanide at 5-carbon. The product of this reaction is a hydroxy ester compound according to formula V:

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII, and R ¹ is lower alkoxycarbonyl or hydroxycarbonyl.

The metal alkoxide used in the reaction corresponds to the formula R ¹⁰ OM, where M is an alkali metal, and R ¹⁰ O- corresponds to the alkoxy substituent R ¹ . The yields of this reaction are most satisfactory if the metal alkoxide is potassium methoxide or sodium methoxide, but other lower alkoxides can be used. Most preferred is potassium alkoxide. Phenoxides, other aryl oxides, as well as aryl sulfides can also be used. The reaction is usually carried out in the presence of an alcohol according to the formula R ¹⁰ OH, where R ^{10 is as} defined above. Other suitable solvents may be used. The substrate of formula VI is preferably present in an amount of from about 2% to about 12% by weight, and more preferably at least about 6% by weight. R ¹⁰ OM is preferably present in a ratio of from about 0.5 to about 4 mol per mol of substrate, more preferably from about 1 to about 2 mol per mol of substrate, and even more preferably about 1.6 mol per mol of substrate. Temperature does not play a decisive role, but the reaction at elevated temperature has a higher productivity. The reaction time is usually from about 4 to about 24 hours, and preferably from about 4 to 16 hours. Typically, the reaction is carried out under reflux, depending on the solvent used.

The time required to achieve equilibrium of the reaction depends on the amount of alkoxide that is added to the reaction mixture and on the method of its addition. The alkoxide can be added in one portion or in several portions, or it can be added continuously. If the alkoxide is added in several portions, it is preferred that approximately 1.6 equivalents of potassium methoxide be added in two stages. With this two-step addition, 1 equivalent of potassium methoxide is first added to the reaction mixture, and then, after 90 minutes, another 0.6 equivalent of potassium methoxide is added. This two-stage addition procedure reduces the equilibrium time compared to adding 1.6 equivalents of sodium methoxide in one serving.

Since equilibrium is a more favorable condition for the production of a hydroxy ester at low diketone concentrations, the reaction is preferably carried out at a rather high degree of dilution, for example up to 40: 1 for the reaction using sodium methoxide. It has been found that significantly higher productivity can be obtained by using potassium methoxide instead of sodium methoxide, since in order to minimize the degree of reverse cyanidation, where the potassium methoxide is the reagent, dilution in the range of about 20: 1 is basically sufficient.

In accordance with the present invention, it has further been found that the reverse cyanidation reaction can be inhibited by using appropriate chemical or physical methods to remove the cyanide ion as a by-product from the reaction zone. Thus, in another embodiment of the present invention, the reaction of the diketone with an alkali metal alkoxide can be carried out in the presence of a precipitating agent for a cyanide ion, such as, for example, a salt containing a cation that forms an insoluble cyanide compound. Such salts may be, for example, zinc iodide, iron sulfate (3) or, basically, any halide, sulfate or other alkaline earth or transition metal salt that is more soluble than the corresponding cyanide. If zinc iodide is present in a ratio of the order of about one equivalent per equivalent of diketone substrate, then it was found that the reaction productivity is significantly increased compared to the reaction carried out in the absence of an alkali metal halide.

Even if a precipitating agent is used to remove the cyanide ion, it remains preferable to carry out the reaction at a sufficiently high degree of dilution, however, when using this precipitating agent, the solvent: diketone substrate molar ratio can be significantly reduced compared to reactions where such an agent is absent. The isolation of the hydroxy ester of formula V can be carried out by methods either involving or not involving extraction, as described below.

To stimulate the production of a hydroxy ester of formula V, the equilibrium of the reaction can also be controlled by removing this hydroxy ester from the reaction mixture after synthesis. Hydroxy ester removal can be carried out stepwise or continuously in a manner such as filtration. Hydroxy ester removal can be used to control the equilibrium of the reaction, either alone or in combination with chemical or physical removal of cyanide from the reaction mixture. Subsequent heating of the obtained filtrate leads to a shift in the equilibrium of the reaction system in the direction of conversion of the remaining diketone of formula VI into a hydroxy ester of formula V.

When the diketone of formula VI was converted to the hydroxy ester of formula V in the crude product, the presence of 5-cyanohydroxy ester was observed in small amounts, usually comprising less than about 5% by weight. It has been suggested that the 5-cyanohydroxy ester is an equilibrium intermediate between a diketone of formula VI and a hydroxy ester of formula V. It has also been suggested that this equilibrium intermediate is formed from diketone by exposure to a 5,7-oxo group of methoxide and protonating the enolate to and from the hydroxy ester by attaching a cyanide by-product ion to the functional 3-keto-Δ ^4,5 -group of the hydroxy ester via the Michael reaction.

In addition, 5-cyano-7-acid and 17-hydroxy-ester of formula V were observed in the crude product by chromatography. It was suggested that the 5-cyano-hydroxy-ester intermediate reacts with the cyanide byproduct ion (present as a result of the decyanation reaction) which introduces a double Δ ^4,5- bond) with the production of 5-cyano-7-acid. It has been suggested that exposure to the cyanide ion leads to dealkylation of the 7-ester group of the 5-cyano-hydroxyether to form 5-cyano-7-acid and the corresponding alkyl nitrile.

In addition, it has been suggested that an unstable intermediate, 17-alkoxide, is formed by the action of methoxide on the 17-spirolactone of the hydroxy ester (or the preceding intermediate, which is then converted to the hydroxy ester). 17-Alkoxide is readily converted to the hydroxy ester after treatment with acid. Therefore, it is usually not found in the matrix product.

5-cyano-hydroxyether, 5-cyano-7-acid and 17-alkoxide are new compounds that can be used as chromatographic markers and as intermediates for the preparation of a hydroxyester. They can be isolated from the crude product in this step of Scheme 1 synthesis. Alternatively, they can be synthesized directly for use as markers or intermediates. 5-Cyano-hydroxyether can be synthesized by reacting a solution of an isolated diketone of formula VI with a base such as an alkoxide or an amine, followed by isolation of the resulting precipitate. A preferred compound is 17-methylhydro-5β-cyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone.

5-Cyano-7-carboxylic acid can be synthesized directly by reacting a diketone of formula VI with a weak aqueous base, such as sodium acetate or sodium bicarbonate, followed by isolation of the resulting residue. The resulting compound is preferably 5-β-cyano-11-α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone.

17-Alkoxide can be synthesized directly by reacting a solution of a hydroxy ester of formula V with an alkoxide to give a mixture of 17-alkoxide and the corresponding hydroxy ester. The resulting compound is preferably dimethyl-11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone.

Preferably, the diketone substrate of formula VI corresponds to formula VIA

and the hydroxy ester product corresponds to formula VA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA, and R ^{1 is} defined in formula V. Preferably, R ³ is hydrogen.

The products of formula V are new compounds that are mainly used as intermediates for the preparation of compounds of formula I, and especially of formula IA. Preferably, the compounds of formula V correspond to formula VA, where —A — A—, —B — B— are —CH ₂ —CH ₂ -, R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ taken together represent a 20-spiroxane ring:

The most preferred compound of formula V is methylhydro-11α, 17α-dihydroxy-3-oxo-pregn-4-en-7α, 21-dicarboxylate, γ-lactone.

The compound of formula V can be isolated by filtration or by acidifying the reaction solution, for example, with a mineral acid, such as aqueous HCl or sulfuric acid, followed by cooling to room temperature and extracting the product with an organic solvent, such as methylene chloride or ethyl acetate. The extract was washed with an aqueous alkaline wash, dried and filtered, after which the solvent was removed. Alternatively, the reaction solution containing the product of formula V may be quenched with concentrated acid. The product solution is concentrated, cooled to a temperature of from about 0 ° C to 25 ° C and the solid product is isolated by filtration.

In a preferred embodiment, methanol and HCN are removed after the completion of the reaction period by distillation, while mineral acid (such as hydrochloric acid or sulfuric acid) is added before distillation, and water is added after distillation. Mineral acid can be added in whole in one portion, stepwise or continuously. In a preferred embodiment, the mineral acid is added continuously over a period of time from about 10 to about 40 minutes, and more preferably from about 15 to about 30 minutes. Similarly, water may be added to the still bottoms in one portion, stepwise or continuously. In a preferred embodiment, the concentrated reaction mixture is cooled, and then water is added after refluxing. Before adding water, this mixture is preferably cooled to a temperature in the range of from about 50 ° C to about 70 ° C, preferably from about 60 ° C to about 70 ° C, and more preferably to about 65 ° C. Water is then added, preferably continuously over a period of time from about 15 minutes to about 3 hours, and more preferably from about 60 minutes to about 90 minutes, maintaining an approximately constant temperature. The product of formula V begins to crystallize from the bottom residue as water is added. After adding water to the mixture, the diluted reaction mixture is maintained at the same temperature for about 1 hour, and then cooled to about 15 ° C for an additional period of time from about 4 to about 5 hours. This mixture is maintained at a temperature of about 15 ° C for a period of time from about 1 to 2 hours. Holding the mixture at 15 ° C for a longer period of time leads to an increase in the yield of the cyano ester in the mixture. This isolation method provides a high quality crystalline product without extraction procedures.

According to another preferred method for isolating a product of formula V, methanol and HCN are removed after the reaction time by distillation, with water and acid being added before the distillation or during the distillation process. Adding water before distillation simplifies the operation, and its gradual addition during the distillation process allows you to maintain, basically, a constant volume in the cube. The product of formula V crystallizes from the bottom residue during the distillation process. This isolation method provides a high quality crystalline product without extraction procedures.

According to another alternative, the reaction solution containing the product of formula V can be quenched by the addition of a mineral acid, for example 4 N HCl, after which the solvent is removed by distillation. Solvent removal is also effective in removing residual HCN from the reaction product. It has been found that for the purification of a compound of formula V, where the compound of formula V serves as an intermediate in the process for preparing epoxymexrenone described above, it is not necessary to carry out multiple solvent extraction. In fact, often such extractions can be completely ruled out. In the case when solvent extraction is used for cleaning, it is desirable to additionally carry out washing with brine and alkaline washing. But in the case when solvent extraction is not carried out, then washing with brine and alkaline washing is also unnecessary. The exclusion of extraction and washing procedures mainly helps to increase the productivity of this process, while not having an adverse effect on the yield and quality of the product, and also avoids the need to dry the washed solution using a desiccant such as sodium sulfate.

The crude 11α-hydroxy-7α-alkoxycarbonyl product is again dissolved in a solvent to carry out the next reaction step of the process, which is the conversion of the 11-hydroxy group to a leaving group at the 11-position, whereby a compound of formula IV is obtained:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above in formula XIII; R ^{1 is} defined in formula V, and R ² is lower arylsulfonyloxy, alkylsulfonyloxy, acyloxy or halide. Preferably, 11α-hydroxy is esterified by reaction with a lower alkyl sulfonyl halide, an acyl halide or an acid anhydride, which is added to a solution containing an intermediate of Formula V. Lower acid anhydrides such as acetic anhydrides and trihalogenated anhydrides such as trifluoroacetic anhydride used to obtain the corresponding leaving acyloxy groups. However, lower alkylsulfonic acid halides are preferred, and methanesulfonyl chloride is particularly preferred. Alternatively, the 11α-hydroxy group can be converted to a halide by reaction of a suitable reagent such as thionyl bromide, thionyl chloride, sulfuryl chloride or oxalyl chloride. Other reagents for the formation of 11α-sulfonic acid esters are tosyl chloride, benzenesulfonyl chloride and trifluoromethanesulfonic anhydride. This reaction is carried out in a solvent containing a halogen hydrogen acceptor, such as triethylamine or pyridine. Inorganic bases such as potassium carbonate or sodium carbonate may also be used. The starting concentration of the hydroxy ester of formula V is preferably from about 5% to about 50% by weight. The esterifying reagent is preferably present in a slight excess. A particularly suitable solvent for this reaction is methylene chloride, but other solvents such as dichloroethane, pyridine, chloroform, methyl ethyl ketone, dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers, acetonitrile, toluene and tetrahydrofuran can also be used. The reaction temperature is mainly determined by the volatility of the solvent. In the case of methylene chloride, the reaction temperature is preferably from about −10 ° C. to about 10 ° C.

The hydroxy ester substrate of formula V preferably corresponds to formula VA:

and the product meets the formula IVA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X is defined in formula XIIIA, R ¹ is lower alkoxycarbonyl or hydroxycarbonyl, and R ^{2 is} defined in formula IV. Preferably, R ³ is hydrogen.

The products of formula IV are new compounds that are used mainly as intermediates for the preparation of compounds of formula I, and especially compounds of formula IA. Preferably, the compounds of formula V correspond to formula VA, where —A — A—, —B — B— represent —CH ₂ —CH ₂ -, R ³ represents hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together represent 20 Spiroxane ring;

A more preferred compound of formula IV is methylhydro-17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxo-pregn-4-en-7α, 21-dicarboxylate, γ-lactone. If the presence of a leaving acyloxy group is desired, then the compound of formula IV is preferably 7-methylhydro-17-hydroxy-3-oxo-11α- (2,2,2-trifluoro-1-oxoethoxy) -17α-pregn-4- en-7α, 21-dicarboxylate, γ-lactone; or 7-methyl-11α- (acetyloxy) -17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone.

If necessary, the compound of formula IV can be isolated by removing the solvent. In this case, it is preferable to rinse the reaction solution first with an aqueous alkaline wash solution, for example 0.5-2 N NaOH, and then rinse with an acid, for example 0.5-2 N HCl. After removal of the reaction solvent, the product is recrystallized, for example, by dissolving the product in methylene chloride, followed by the addition of another solvent, such as ethyl ether, which reduces the solubility of the product of formula IV, which leads to its precipitation in crystalline form.

By isolating a product of formula IV or by preparing a reaction solution for converting an intermediate of formula IV to an intermediate of formula II, as described below, all extraction and / or washing steps can be omitted, and instead, the solution can be treated with ion-exchange resins to remove acidic and basic impurities. This solution is first treated with an anion exchange resin, and then with a cation exchange resin. Alternatively, the reaction solution may be first treated with inorganic adsorbents, such as basic alumina or basic silica, followed by washing with dilute acid. Basic silica or basic alumina can be mainly mixed with the reaction solution in a ratio of from about 5 to about 50 g per kg of product, and preferably from about 15 to about 20 g per kg of product. In the case of ion-exchange resins or inorganic adsorbents, the treatment can be carried out simply by suspending the resin or inorganic adsorbent with a reaction solution with stirring at room temperature and then removing the resin or inorganic adsorbent by filtration.

In an alternative and preferred embodiment of the present invention, the resulting compound of formula IV is isolated in crude form as a concentrated solution by removing part of the solvent. This concentrated solution is directly used in the next process step, in which the leaving 11α group is removed from the compound of formula IV, whereby an ester of formula II is obtained:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined above in formula XIII, and R ^{1 is} defined in formula V. To carry out this reaction, the substituent R ^{2 of the} compound of formula IV can be any leaving a group whose removal is effective for the formation of a double bond between 9- and 11-carbons. The leaving group is preferably a lower alkylsulfonyloxy or acyloxy substituent which is removed by reaction with an acid or alkali metal salt. Mineral acids may be used for this, but lower alkanoic acids are preferred. In addition, a preferred reagent for this reaction is an alkali metal salt and an alkanoic acid used. It is particularly preferred that the leaving group contains mesyloxy and the reagent for this reaction contains formic acid or acetic acid, or an alkali metal salt of one of these acids or another lower alkanoic acid. If the leaving group is mesyloxy, and the reagent removed is either acetic acid and sodium acetate, or formic acid and potassium formate, a relatively high ratio of 9.11-olefin to 11.12-olefin is achieved. If free water is present during the removal of the leaving group, then there is a tendency to the formation of impurities, in particular, 7.9-lactone

(where: -A-A-, R ³ , -B-B-, R ⁸ and R ^{9 are} defined above in formula XIII), which are difficult to remove from the final product. Therefore, to remove water present in formic acid, acetic anhydride or another desiccant is used. The content of free water in the reaction mixture must be kept below about 0.5%, preferably below about 0.1% by weight, as determined by Karl Fischer analysis for water, based on the weight of the total reaction solution, before carrying out the reaction. Although it is preferable that the reaction mixture is kept practically dry, good results can also be obtained with a water content of 0.3% by weight. It is preferable that the boot reaction mixture contains from about 4% to about 50% of the mass. a substrate of formula IV in alkanoic acid. Preferably, this mixture contains from about 4% to about 20% of the mass. alkali metal salts with acid. If acetic anhydride is used as a desiccant, it is preferred that it is present in a ratio of from about 0.05 mol to about 0.2 mol per mole of alkanoic acid.

It was found that the amounts of the by-product of 7,9-lactone and 11,12-olefin in the reaction mixture would be relatively small if the elimination reagent contains a combination of trifluoroacetic acid, trifluoroacetic anhydride and potassium acetate, which is used as a reagent to eliminate the leaving group and formation of a complex ether (9,11-olefin). Trifluoroacetic anhydride serves as a drying agent and should be present in an amount of at least about 3% by weight, more preferably at least about 15% by weight, and most preferably about 20% by weight. based on the weight of the eliminating reagent containing trifluoroacetic acid.

In this stage of Synthesis Scheme 1, in addition to 7.9-lactone, there are other impurities and by-products that can be used as intermediates for synthesis and as chromatographic markers. The new 4,9,13-triene of the ester of formula II (e.g., 7-methylhydro-17-methyl-3-oxo-18-norpregna-4.9 (11), 13-triene-7α, 21-dicarboxylate) was isolated from product solution by chromatography. Obviously, the produced amount of this compound increases with increasing reaction time for this synthesis step. It has been suggested that this compound is formed by protonation of the lactone and the resulting C17 carbonium ion promotes the migration of the angular methyl group from the C13 position. The deprotonation of this intermediate leads to the formation of 4,9,13-triene.

The new 5-cyano-Δ ^11,12 -compound of the ester of formula II (for example, 7-methylhydro-5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ- lactone) and a new 5-cyano-ester compound of the formula II (for example, 7-methyl-hydro-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ- lactone) were also isolated from the crude product by chromatography. It has been suggested that these compounds are formed by dehydration of the residual 5-cyano-7-acid and 5-cyano-hydroxyether, respectively, which are present in the solution of the crude product obtained in the third stage of Synthesis Scheme 1.

The new C17 epimer of the ester of formula II (for example, 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone) was also isolated from crude product by chromatography. It has been suggested that the acidic conditions of the elimination reaction can lead to racemization of the chiral C17 center to produce the 17-epimer of the complex ester. The 17-epimer can be synthesized directly by reacting a compound of formula IV with a solution of potassium formate, formic acid and acetic anhydride, followed by isolation of the 17-epimer.

Although no impurities were observed in the crude product solution, the 11-hydroxy ester of formula V can be prepared by oxidizing the 11-hydroxy of the corresponding hydroxy ester with a suitable oxidizing agent such as Jones reagent. The obtained 11-ketone is preferably 7-methylhydro-17-hydroxy-3,11-dioxo-17α-pregna-4-en-7α, 21-dicarboxylate, γ-lactone.

Alternatively, the leaving 11α groups of the compound of formula IV can be removed to give an ester of formula II by heating a solution of formula IV in an organic solvent such as DMSO, DMF or DMA.

In addition, in accordance with the present invention, the compound of formula IV is first reacted with an alkenyl alkanoate such as isopropenyl acetate in the presence of an acid such as toluenesulfonic acid or an anhydrous mineral acid such as sulfuric acid to form a 3-enol ester

compounds of formula IV. Alternatively, 3-enol ester can be prepared by treating a compound of formula IV with an acid anhydride and a base such as acetic acid and sodium acetate. Other alternative methods are to treat a compound of formula IV with ketene in the presence of an acid to give a compound of formula IV (Z). The intermediate of formula IV (Z) is then reacted with an alkali metal formate or acetate in the presence of formic or acetic acid to obtain Δ ^9,11- enol acetate of formula IV (Y):

which can then be converted to an ester of formula II in an organic solvent, preferably an alcohol, such as methanol, either by thermal decomposition of the enol acetate or by reaction with an alkali metal alkoxide. The elimination reaction is highly selective for the ester of formula II, with 11,12-olefin and 7,9-lactone being preferred and this selectivity is maintained during the conversion of enol acetate to enone.

Preferably, the substrate of formula IV corresponds to formula IVA:

and the product corresponds to the formula IIA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA, R ^{1 is} defined in formula V, and R ³ is preferably hydrogen.

If necessary, the compound of the formula can be isolated by removing the solvent, dissolving the solid in cold water and extracting with an organic solvent such as ethyl acetate. After appropriate washing and drying steps, the product is isolated by removing the extraction solvent. The ester is then dissolved in a solvent suitable for conversion to the Formula I product. Alternatively, the ester can be isolated by adding water to the concentrated product solution and filtering the solid product, followed by the preferred removal of 7.9-lactone. The conversion of the substrate of formula II to the product of formula IA can be carried out by the method described in US patent No. 4559332, which in its entirety is introduced into the present invention by reference, or more preferably using a new reaction using a halogenacetamide stimulator, as described below.

In another embodiment of the present invention, a hydroxy ester of formula V can be converted to an ester of formula II without isolation of an intermediate of formula IV. In this method, the hydroxy ester is dissolved in an organic solvent such as methylene chloride; and either an acylating agent, for example methanesulfonyl chloride, or a halogenating agent, for example sulfuryl chloride, is added to the solution. The resulting mixture is stirred and, if halogenation is provided, an HCl acceptor such as imidazole is added. This reaction is highly exothermic and, therefore, it must be carried out at an adjustable speed with complete cooling. After adding the base, the resulting mixture is heated to a moderate temperature, for example from about 0 ° C to room temperature or slightly higher, and the reaction is usually carried out over a period of time from about 1 hour to about 4 hours. After completion of the reaction, the solvent is removed, preferably under high vacuum (for example, from about 24 "to about 28" Hg (inch Hg), from about -10 ° C to about + 15 ° C, more preferably from about 0 to about 5 ° C, to concentrate the solution and remove excess base. Then, to be converted to an ester, the substrate is redissolved in an organic solvent, preferably in a halogenated solvent, such as methylene chloride.

The reagent for eliminating the leaving group is preferably prepared by mixing an organic acid, an organic acid salt and a drying agent, preferably formic acid, alkali metal formate and acetic anhydride, respectively, in a dry reactor. The addition of acetic anhydride is accompanied by the release of heat and leads to the release of CO, and therefore the rate of addition must be appropriately regulated. To stimulate the removal of water, the temperature of this reaction is preferably maintained in the range of from about 60 to about 90 ° C, and most preferably from about 65 to about 75 ° C. Then, the indicated reagent is added to the solution of the product of compound IV for the elimination reaction. After a period of time from about 4 to about 8 hours, the reaction mixture is preferably heated to a temperature of at least about 85 ° C, but preferably not higher than about 95 ° C, until the volatile distillate is completely removed, and then further heated until the reaction is complete, usually within about 1 to about 4 hours. The reaction mixture is cooled and, after isolation by standard extraction methods, this ester can be isolated, if necessary, by evaporation of the solvent.

In addition, it was found that the ester of formula II can be isolated from the reaction solution by an alternative method, which avoids the need for extraction steps after the elimination reaction, which makes it possible to reduce material costs, increase yield and / or increase productivity. In this method, the ester is precipitated by diluting the reaction mixture with water after removing formic acid. Then the product is isolated by filtration. In this case, no extraction is required.

According to another alternative method for converting a compound of formula V to an ester of formula II without isolating a compound of formula IV, the 11α-hydroxy group of the hydroxy ester of formula V is replaced with halogen, after which the ester of formula II is formed in situ due to thermal dehydrohalogenation. The replacement of the hydroxy group with a halogen is carried out by reaction with a sulfuryl halide, preferably sulfuric chloride, while cooling in the presence of an acceptor of a hydrogen halide such as imidazole. The hydroxy ester is dissolved in a solvent such as tetrahydrofuran and cooled to a temperature of from about 0 ° C to about -70 ° C. Sulfuryl chloride is then added and the reaction mixture is heated to a moderate temperature, for example to room temperature, for a period of time sufficient to complete the elimination reaction, usually for a period of time from about 1 to about 4 hours. The method of implementing this embodiment of the invention not only allows combining the two steps into one, but also avoids the use of: a halogenated reaction solvent; an acid (such as acetic acid) and a desiccant (such as acetic anhydride or sodium sulfate). In addition, this reaction does not require reflux conditions and avoids the production of a CO by-product resulting from the use of acetic acid as a drying agent.

According to a particularly preferred embodiment of the present invention, the diketone compound of formula VI can be converted to epoxymexrenone or another compound of formula I without isolating any intermediate in purified form. According to this preferred method, the reaction solution containing the hydroxy ester is quenched with a strong acid solution, cooled to room temperature, and then extracted with an appropriate solvent for extraction. Before extraction, an aqueous solution of an inorganic salt, for example about 10% by weight, is preferably added to the reaction mixture. saturated saline. The extract is washed and dried by azeotropic distillation to remove the methanol solvent remaining after the ketone cleavage reaction.

Then, the resulting concentrated solution containing from about 5% to about 50% of the mass. the compounds of formula V are cold contacted with an acylating or alkylsulfonating reagent to form a sulfonic acid ester or dicarboxylic acid ester. After completion of the alkyl sulfonation or carboxylation reaction, the reaction solution is passed through an acid column and then with a basic ion exchange resin to remove the main and acid impurities. After each passage, the column is washed with an appropriate solvent, for example methylene chloride, to recover the residual sulfonic or dicarboxylic acid ester from it. The combined eluate and washings are combined and concentrated, preferably in vacuo, to give a concentrated solution containing a sulfonic or dicarboxylic acid ester of formula IV. This concentrated solution is then contacted with a dry reagent containing an agent effective to remove the leaving 11α-ester group and to remove hydrogen to form a 9.11 double bond. Preferably, the leaving group removal reagent comprises a solution of a dry reagent, formic acid / alkali metal formate / acetic anhydride described above. After completion of the reaction, the reaction mixture is cooled, and formic acid and / or other volatile components are removed in vacuo. The residue was cooled to room temperature, subjected to the appropriate washing steps, and then dried, resulting in a concentrated solution containing an ester of formula II. This ester can then be converted to epoxymexrenone or another compound of formula I using the method described above or the method described in US Pat. No. 4,559,332.

In a particularly preferred embodiment, the solvent is removed from the reaction solution under vacuum and the product of formula IV is partitioned between water and an appropriate organic solvent, for example ethyl acetate. The aqueous layer is then back extracted with an organic solvent and this back extract is washed with an alkaline solution, preferably an alkali metal hydroxide solution containing an alkali metal halide. The organic phase is preferably concentrated in vacuo to give the ester product of formula II. This product of formula II can then be dissolved in an organic solvent, for example methylene chloride, and then it can be reacted by the method described in patent 322 to obtain a product of formula I.

When trihaloacetonitrile is used in the epoxidation reaction, it has been found that the choice of solvent is very important, with halogenated solvents being highly preferred, and methylene chloride being particularly preferred. Solvents such as dichloroethane and chlorobenzene give a quite sufficient yield, but a reaction medium containing methylene chloride gives a much better yield. Solvents such as acetonitrile and ethyl acetate generally give insufficient yield, and the reaction in solvents such as methanol or water / tetrahydrofuran gives a small amount of the desired product.

In addition, in accordance with the present invention, it was found that a significant improvement in the synthesis of epoxymexrenone can be realized by using trihaloacetamide instead of trihaloacetonitrile as a peroxide activator for the epoxidation reaction. In a particularly preferred method, this epoxidation is carried out by reacting a substrate of formula IIA with hydrogen peroxide in the presence of trichloroacetamide and an appropriate buffer. This reaction is preferably carried out at a pH in the range of from about 3 to about 7, and most preferably from about 5 to about 7. However, despite this, this reaction can be successfully carried out at a pH having values outside these preferred ranges.

The most suitable results are obtained using a buffer containing dipotassium biphosphate and / or using a buffer containing a combination of dipotassium biphosphate and potassium bisphosphate in appropriate ratios from about 1: 4 to about 2: 1, and most preferably in the range of about 2: 3. Borate buffers can also be used, but they usually give slower conversion reactions than potassium bisphosphate or mixtures of K ₂ HPO ₄ / KH ₂ PO ₄ . Regardless of the buffer used, it should provide a pH in the range specified above. Regardless of the full composition of the buffer or the accuracy of the pH that it can provide, it has been observed that the reaction proceeds much more efficiently if at least part of the buffer contains a dibasic bisphosphate ion. Obviously, this ion can mainly precipitate as a homogeneous catalyst during the formation of an adduct or complex containing a stimulant and hydroxyperoxide ion, the production of which, in turn, can play an important role in the mechanism of the epoxidation reaction. Thus, only a small concentration of catalyst can be a quantitative requirement for a dibasic bisphosphate (preferably starting from K ₂ HPO ₄ ). In general, it is preferred that K ₂ HPO ₄ be present in an amount of at least about 0.1 equivalents, for example from about 0.1 to about 0.3 equivalents per equivalent of substrate.

This reaction is carried out in a suitable solvent, preferably methylene chloride, but alternatively other halogenated solvents such as chlorobenzene or dichloroethane can also be used. It was found that satisfactory results also give toluene and a mixture of toluene and acetonitrile. Without claiming to be a specific theory, it should be noted that the reaction proceeds more efficiently in a two-phase system in which the resulting hydroperoxide intermediate is distributed over the organic phase with a low water content and reacts with the substrate in the organic phase. Thus, preferred solvents are those in which the solubility in water is low. Effective isolation from toluene is stimulated by the inclusion of another solvent, such as acetonitrile.

When converting the substrates of the formula II into the products of the formula I, the use of toluene is advantageous, since these substrates are readily soluble in toluene, in contrast to these products. Thus, the product precipitates during the reaction when the conversion reaches 40-50%, which leads to the production of a three-phase mixture from which this product can be easily isolated by filtration. When carrying out the conversion reaction at this stage of the process, methanol, ethyl, acetonitrile, taken separately, and THF and THF / water are not as effective as halogenated solvents or toluene.

Although trichloroacetamide is a highly preferred reagent, other trihaloacetamides such as trifluoroacetamide and chlorodifluoroacetamide can also be used. Trihalogenbenzamide and other compounds having an arylene, alkenyl or alkynyl group (or another group that promotes the transfer of the electron-accepting action of the electron-acceptor group to the carbonyl amide) between the electron-acceptor trihalogenomethyl group and the amide carbonyl can also be used. Heptafluorobutyramides may also be used, but with less favorable results. Typically, the peroxide activator may correspond to the formula:

R ° C (O) NH ₂

where R ° represents a group having at least the same high electron-acceptor force (as measured by the sigma constant) as the monochloromethyl group. For maximum efficiency, the electron-acceptor group is preferably attached directly to the amide carbonyl. More specifically, the peroxide activator may correspond to the formula

where R ^P represents a group that allows you to transfer the electron-accepting effect of the electron-acceptor group on the carbonyl amide and which is preferably selected from arylene, alkenyl, alkynyl and - (CX ⁴ X ⁵ ) _n groups; X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} independently selected from halogen, hydrogen, alkyl, halogen alkyl, cyano and cyanoalkyl; and n is 0, 1 or 2; provided that if n = 0, then at least one of X ¹ , X ² and X ³ represents halogen; and if R ^P represents - (CX ⁴ X ⁵ ) _n and n = 1 or 2, then at least one of X ⁴ and X ⁵ represents halogen. If any of X ¹ , X ² , X ³ , X ⁴ and X ^{5 is} not halogen, then it is preferably halogenated, and more preferably perhalogenated. Particularly preferred activators are those activators in which n = 0, and at least two of X ¹ , X ² and X ³ are halogen; or such activators in which R ^P represents - (CX ⁴ X ⁵ ) _n ; n is 1 or 2; at least one of X ⁴ and X ⁵ is halogen, and the other of X ⁴ and X ⁵ are halogen or perhaloalkyl; and X ¹ , X ² and X ³ are halogen or perhaloalkyl. Each of X ¹ , X ² , X ³ , X ⁴ and X ^{5 is} preferably Cl or F, and most preferably Cl, although mixed halides, i.e. perchloralkyl or perbromoalkyl and combinations thereof, can also be used, provided that carbon, directly bonded to the amide carbonyl, is substituted by at least one halogen group.

Preferably, the peroxide activator is present in a ratio of at least about 1 equivalent, more preferably from about 1.5 to about 2 equivalents, per equivalent of the initially present substrate. Hydrogen peroxide should be loaded into the reaction at least in moderate excess, or it should be constantly added as the epoxidation reaction proceeds. Although the reaction absorbs only one to two equivalents of hydrogen peroxide per mole of substrate, it is preferable that hydrogen peroxide be loaded in a significant excess with respect to the originally present substrate and activator. Without claiming to be a specific theory, it should be noted that the reaction mechanism provides for the formation of an activator adduct and peroxide anion and that this reaction is reversible with an equilibrium shift towards the reverse reaction and that therefore it is necessary to use a significant initial excess of hydrogen peroxide to undergo a direct reaction direction. The exact reaction temperature is not critical and this reaction can be effectively carried out at a temperature ranging from about 0 ° C to about 100 ° C. The optimum temperature depends on the choice of solvent. In general, a preferred temperature is from about 20 ° C. to about 30 ° C., but with certain solvents, for example in toluene, this reaction can be advantageously carried out at a temperature of from about 60 ° C. to about 70 ° C. At a temperature of about 25 ° C., the reaction usually takes less than about 10 hours, generally from about 3 to about 6 hours. If necessary, at the end of the reaction cycle, to achieve complete conversion of the substrate, an additional amount of activator and hydrogen peroxide can be added.

At the end of the reaction cycle, the aqueous phase is removed, the organic reaction solution is preferably washed to remove water-soluble impurities, after which the product can be isolated by removing the solvent. Before removing the solvent, the reaction solution should be washed with at least mild to moderately alkaline washing, for example sodium carbonate. Preferably, the reaction mixture is washed sequentially: with a weak reducing solution, such as a weak (for example, about 3% by weight) solution of sodium sulfite in water; alkaline solution, for example NaOH or KOH (preferably about 0.5n); an acidic solution such as HCl (preferably about 1N); and a final neutral wash containing water or saline, preferably saturated saline, to minimize product loss. Prior to removal of the reaction solvent, a predominantly other solvent, such as an organic solvent, preferably ethanol, may be added so that this product can be isolated by crystallization after distillation to remove a more volatile reaction solvent.

It should be noted that a new method of epoxidation using trichloroacetamide or another new peroxide activator has an application that goes far beyond the scope of various schemes for the preparation of epoxymexrenone, and in fact can be used to form epoxides via olefinic double bonds in a wide range of substrates subjected to reaction in liquid phase. This reaction is particularly effective in unsaturated compounds in which the olefins are tetrasubstituted and trisubstituted, that is, R ^a R ^b C = CR ^c R ^d and R ^a R ^b C = CR ^c H, where R ^a -R ^d are substituents not being hydrogen. This reaction proceeds most quickly and completely, where the substrate is a cyclic compound with a trisubstituted double bond, or a cyclic or acyclic compound with a tetrasubstituted double bond. Examples of substrates for the epoxidation reaction are Δ ^5.11 -canrenone and the following substrates:

Since the reaction proceeds more quickly and more fully with trisubstituted and tetrasubstituted double bonds, it is especially effective for the selective epoxidation of such double bonds in compounds, which may include other double bonds, where the olefin carbon atoms are monosubstituted or even disubstituted.

Other non-limiting examples illustrating the general epoxidation reaction are the following epoxidation reactions:

In addition, it should be noted that this reaction can be used to predominantly carry out the epoxidation reaction of monosubstituted or even disubstituted double bonds, such as 11,12-olefin in various steroid substrates.

However, since mainly double bonds with a higher degree of substitution, for example 9,11-olefin, with a high degree of selectivity are epoxidized, the method of the present invention is particularly effective for achieving high yields and productivity in the epoxidation steps in various reaction schemes also described in this application.

This improved process has been shown to be particularly advantageous when used to prepare the compound.

by epoxidizing the compound

; и

; and

with the connection

by epoxidizing the compound:

The many advantages of the process of the present invention have been demonstrated in which trichloroacetamide is used instead of trichloroacetonitrile as an oxygen transfer reagent in an epoxidation reaction. The trichloroacetamide reagent system has a low affinity for electron-deficient olefins, such as α, β-unsaturated ketones. This allows selective epoxidation of an olefin with non-conjugated double bonds in a substrate containing both types of double bonds. In addition, in complex substrates such as steroids, disubstituted and trisubstituted olefins can be differentiated by reaction. For example, good selectivity is observed when epoxidizing isomeric Δ-9,11 and Δ-11,12 compounds. In this case, 9,11-epoxide is formed with a minimal reaction of an isomer containing a Δ-11,12-double bond. Accordingly, the yield of the reaction, the profile of the products, and the final purity of the product are generally higher than the reactions in which trihaloacetonitrile is used. In addition, it was found that the significantly excess oxygen production observed with trihaloacetonitrile was minimized with trichloroacetamide, giving greater reliability to this epoxidation process. In addition, in contrast to the reaction stimulated by trichloroacetonitrile, the reaction using trichloroacetamide gives a minimal exothermic effect, which facilitates the regulation of the temperature profile of the reaction. According to observations, it was found that in this reaction the effect of mixing is minimal, and the productivity of the reactor is higher, and this reaction has additional advantages compared to the reaction using trichloroacetonitrile. This reaction is more suitable for large-scale production than the reaction stimulated by trichloroacetonitrile. Isolation of the product and its purification are simple. Bayer-Williger oxidation of the carbonyl functional group (peroxide-stimulated conversion of ketone to ester) was not observed, as was revealed in an experiment using m-chloroperoxybenzoic acid or other peracids. This reagent is inexpensive, readily available and technologically advanced.

In addition, in the crude product obtained in step of Synthesis Scheme 1, in which the ester of formula II was converted to a compound of formula I, the following compounds were observed by chromatography:

(1) the new 11α, 12α-epoxide of the ester of formula II, for example 7-methylhydro-11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone ;

(2) the new 4,5: 9,11-dietepoxide of the ester of formula II, for example 7-methylhydro-4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate , γ-lactone;

(3) a novel 12-ketone ester of formula II, for example 7-methylhydro-17-hydroxy-3,12-dioxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone;

(4) a new 9,11-dihydroxy ester of formula II, for example 7-methylhydro-9α, 11β, 17-trihydroxy-3-oxo-17α-pregna-4-en-7α, 21-dicarboxylate, γ-lactone;

(5) a new 12-hydroxy analogue of the ester of formula II, for example 7-methylhydro-12α, 17-dihydroxy-3-oxo-17α-pregna-4.9 (11) -diene-7α, 21-dicarboxylate, γ- lactone; and

(6) a novel 7-acid of a compound of formula I, for example 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, γ-lactone.

These compounds are used as synthetic intermediates and / or chromatographic markers to prepare a compound of formula I, in particular epoxymexrenone.

It has been suggested that the 11α, 12α-epoxide of the ester of formula II is formed through an impurity produced in the previous step in which the compound of formula IV is converted to the ester of formula II. This impurity was isolated by chromatography and is a complex Δ ^11,12- ether. It is usually produced together with a complex Δ ^9,11- ether in a ratio of about 90:10 (Δ ^9,11- ether: Δ ^11,12- ether), although this ratio may vary. As a result of the oxidation of the Δ ^11,12- ester, the 11α, 12α-epoxide is formed during the conversion of the ester of the formula II to the compound of the formula I.

4,5: 9,11-Diepoxide of the complex ester of formula I is isolated by chromatography. It has been suggested that it is formed as a result of superepoxidation of the complex ester. In the raw product, it is usually present in an amount of about 5% of the mass. or less, although this amount may vary.

The 12-ketone ester of formula II is isolated by chromatography. It has been suggested that it is formed as a result of allylic oxidation of the ester. In the raw product, it is usually present in an amount of about 5% of the mass. or less, although this amount may vary. The level of 12-ketone found in the crude product using trichloroacetonitrile as an activator of hydrogen peroxide was significantly higher than the level detected with trichloroacetamide as an activator.

The 9.11-dihydroxy product of the ester of formula II is isolated by chromatography. In the raw product, it is usually present in an amount of about 5% of the mass. or less, although this amount may vary. It has been suggested that it is formed by hydrolysis of an epoxide of formula I.

The 12-hydroxy product of the ester of formula II is isolated by chromatography. In the raw product, it is usually present in an amount of about 5% of the mass. or less, although this amount may vary. It has been suggested that it is formed by hydrolysis of 11,12-epoxide followed by elimination of 11β-hydroxy.

In addition, the compounds of formula I obtained in accordance with this description can be further modified to produce a metabolite, derivative of a prodrug, or the like, with improved properties (such as increased solubility and absorption), which facilitates the introduction and / or effectiveness epoxymexrenone. 6-Hydroxy compounds of formula I (for example, 7-methylhydro-6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone) are new compounds that have been identified as a possible metabolite in rats. 6-Hydroxy-metabolite can be obtained from the corresponding ethyl ester of enol (for example, 7-methylhydro-9α, 11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-en-7α, 21-dicarboxylate, γ- lactone). Enol ethyl ester of a compound of formula I can be prepared according to the procedure described in R. M. Weier & L. M. Hofmann (J. Med. Chem. 1977, 1304), which is incorporated herein by reference. The enol ethyl ester is then reacted with m-chloroperoxybenzoic acid to give the corresponding 6-hydroxy compound of formula I.

It has further been suggested that the monocarboxylic salts of epoxymexrenone, especially potassium and sodium salts, may serve as an appropriate alternative to facilitate administration of a compound of formula I to an individual for whom administration of an aldosterone antagonist is indicated. Under mild basic conditions, it is possible to selectively open the spirolactone of the compounds of formula I without hydrolysis of the C7 ester substituent to give the corresponding 17β-hydroxy-17α- (3-propionic acid) analog. These open-circuit analogs are more polar than their lactone analogs and have a shorter retention time when analyzed by reverse phase HPLC. Acid conditions usually lead to the regeneration of the lactone ring.

Under more severe conditions, spirolactone opens and complex C7 hydrolyzes to form the corresponding by-products, 7β-hydroxy-17α (3-propionic acid) -7-acid analogs of the compounds of formula I. These dicarboxylic acids have a shorter retention time when they are analyzed using reverse -phase HPLC. Acidic conditions (for example, treatment with a dilute acid such as 0.1-4 M hydrochloric acid) usually result in regeneration of the lactone ring of the dicarboxylic acid.

The new epoxidation process of the present invention is highly effective as a final step in Synthesis Scheme 1. In a particularly preferred embodiment of the invention, the entire process of Scheme 1 proceeds as follows:

Scheme 2.

The second scheme of the new reactions of the present invention (Scheme 2) begins with the use of canrenone or another substrate of the corresponding formula XIII

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above in formula XIII.

In the first step of this process, a substrate of formula XIII is converted to a product of formula XII

using a cyanidation reaction scheme essentially the same as described above for converting a substrate of formula VIII to an intermediate of formula VII. Preferably, the substrate of formula XIII corresponds to formula XIIIA:

and enamine product corresponds to the formula XIIA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA. Preferably, R ³ is hydrogen.

In a second step of Scheme 2, an enamine of formula XII is hydrolyzed to give an intermediate diketone product of formula XI:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are as} defined above in formula XIII using a reaction scheme substantially similar to that described above for converting a substrate of formula VIII to an intermediate of formula VII . Preferably, the substrate of formula XII corresponds to formula XIIA:

and the diketone product corresponds to the formula XIA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA. Preferably, R ³ is hydrogen.

In addition, in accordance with reaction scheme 2, the diketone of formula XI is reacted with an alkali metal alkoxide to form mexrenone or another product corresponding to formula X,

where each of -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII, and R ^{1 is} defined in formula V.

This method is carried out using mainly the reaction scheme described above for the conversion of compounds of formula VI to compounds of formula V. Preferably, the substrate of formula XI corresponds to formula XIA

and the intermediate product corresponds to the formula XA:

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X are defined in formula XIIIA, and R ^{1 is} defined in formula V. Preferably, R ³ is hydrogen.

Then, mexrenone and other compounds of formula X are subjected to 9α-hydroxylation using a new biological conversion method and the products of formula IX are obtained:

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined above in formula XIII, and R ^{1 is} defined in formula V.

The microorganisms that can be used in this hydroxylation step are; Nocardia conicruria ATCC 31548, Nocardia aurentia ATCC 12674, Corynespora cassiicola ATCC 16718, Streptomyces hydroscopicus ATCC 27438, Mortierella isabellina ATCC 42613, Beauvria bassiana ATCC 7519, Penicillum purpurogenum ATCC 46581, Hypomyces chrysospermus IMI 109891, Thamnostylum piriforme ATCC 8992, Cunninghamella blakesleeana ATCC 8688a, Cunninghamella echinulata ATCC 3655, Cunninghamella elegans ATCC 9245, Trichothecium roseum ATCC 12543, Epicoccum Humicola ATCC 12722, Saccharopolyspora eythrae ATCC 11635, Beauvria bassiana ATCC 13144, Arthrobacter simplex, Bacteriumrdoclodlocclooxyloxy ATCC 14887, Pseudomonas testosteroni ATCC 11996, Rhodococcus egui ATCC 21329, Mycobacterium fortuitum NRRL B8119 and Rhodococcus rhodochrous ATCC 19150. This reaction is carried out mainly by the method described above with reference to FIG. 1 and 2. Particularly preferred is the method illustrated in FIG. 1.

The culture medium, which can be used in biological conversion reactions, preferably contains from about 0.05% to about 5% of the mass. available nitrogen; from about 0.5% to about 5% of the mass. glucose from about 0.25% to about 2.5% of the mass. yeast derivative; and from about 0.05% to about 5% of the mass. available phosphorus. Particularly preferred culture media are the following:

based on soy flour: from about 0.5% to about 3% of the mass. glucose from about 0.1% to about 1% of the mass. soy flour; from about 0.05% to about 5% of the mass. alkali metal halide; and from about 0.05% to about 0.5% of the mass. a yeast derivative such as autolysed yeast or yeast extract; from about 0.05% to about 5% of the mass. a phosphate salt such as K ₂ HPO ₄ ; pH = 7;

peptone-yeast extract-glucose: from about 0.2% to about 2% of the mass. peptone; from about 0.05% to about 0.5% of the mass. yeast extract; and from about 2% to about 5% of the mass. glucose

Meller-Hinton medium: from about 10% to about 40% of the mass. beef broth; from about 0.35% to about 8.75% of the mass. casamino acids; and from about 0.15% to about 0.7% of the mass. starch.

Fungi can be cultured in a culture medium based on soy flour or peptone, while actinomycetes and eubacteria can grow in a medium based on soy flour (to which 0.5% -1% by weight of a carboxylic acid salt, such as sodium formate for biological transformations) or in Meller-Hinton broth.

The preparation of 11β-hydroxymexrenone from mexrenone by fermentation is discussed in Example 19B. Similar biological transformation methods can be used to prepare other starting compounds and intermediates. Example 19A describes the biological conversion of androstenedione to 11β-hydroxyandrostenedione. Example 19C describes the biological conversion of mexrenone to 11α-hydroxymexrenone, Δ ^1,2- mexrenone, 6β-hydroxymexrenone, 12β-hydroxymexrenone and 9α-hydroxymexrenone. Example 19D describes the biological conversion of canrenone to Δ ^9,11 -canrenone.

The products of formula IX are new compounds that can be isolated by filtration, washed with a suitable organic solvent, for example ethyl acetate, and recrystallized from the same or similar solvent. They are valuable, mainly as intermediates for the preparation of compounds of formula I, and especially compounds of formula IA. Preferably, the compounds of formula IX correspond to formula IXA, where -A-A-, -B-B- represent -CH ₂ -CH ₂ -, R ³ represents hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together represent 20 Spiroxane ring:

In the next step of Synthesis Scheme 2, the product of formula IX is reacted with a dehydrating reagent (suitable dehydrating agents such as PhSOCl or ClSO ₃ H are known to those skilled in the art) to give a compound of formula II

where: -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined above in formula XIII, and R ^{1 is} defined in formula V. Preferably, the compound of formula IX corresponds to formula IXA

and the intermediate product corresponds to the formula IIA

where each of -A-A-, -B-B-, R ³ , Y ¹ , Y ² and X is defined in formula XIIIA, and R ^{1 is} defined in formula V. Preferably, R ³ is hydrogen.

In the final step of this synthesis scheme, the product of formula II is converted to the product of formula I by epoxidation by the method described in US Pat. No. 4,559,332; or preferably the novel epoxidation process of the present invention described above.

In a particularly preferred embodiment of the invention, the entire process of Scheme 2 is as follows:

Scheme 3.

In this case, the synthesis begins, starting from the substrate corresponding to the formula XX

where: -A-A- and R ^{3 are} defined above in formula XIII, -B-B- is defined in formula XIII, except that neither R ⁶ nor R ⁷ are part of a ring fused to ring D at 16, 17-positions, and R ²⁶ represents lower alkyl, preferably methyl. Preferably, R ³ is hydrogen. The reaction of the substrate of formula XX with sulfonium ylide forms an epoxy intermediate corresponding to formula XIX:

where: -A-A-, -B-B-, R ³ and R ^{26 are as} defined above in formula XX. R ^{3 is} preferably hydrogen.

In the next step of Synthesis Scheme 3, an intermediate of formula XIX is converted to another intermediate of formula XVIII:

where: -A-A-, -B-B-, R ^{3 are} defined above in formula XX. R ^{3 is} preferably hydrogen. In this step, a substrate of formula XIX is converted to an intermediate of formula XVIII by reaction with NaCH (COOEt) ₂ in the presence of a base in a solvent.

After heat treatment of the compound of formula XVIII and its treatment with water and an alkali metal halide, a decarboxylated intermediate corresponding to formula XVII is obtained:

where: -A-A-, -B-B-, R ^{3 are} defined above in formula XX. R ^{3 is} preferably hydrogen.

This method of converting a compound of formula XX to a compound of formula XVII corresponds generally to the method described in US Pat. Nos. 3,897,417, 3,431,288 and 3,300,489, which are hereby incorporated by reference in their entirety. Although the substrates are different, however, the reagents, mechanisms, and conditions for administering the 17-spirolactone moiety are substantially the same.

The reaction of an intermediate of formula XVII with a dehydrogenating reagent gives another intermediate of formula XVI:

where: -A-A-, -B-B-, R ^{3 are} defined above in formula XX. R ^{3 is} preferably hydrogen.

Commonly used dehydrogenating agents are dichlorodicyanobenzoquinone (DDQ) and chloranil (2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, dehydrogenation can be carried out by subsequent halogenation at the 6-position of carbon, and then through a dehydrohalogenation reaction.

Then, the intermediate of formula XVI is converted to the enamine of formula XVB:

where -A-A-, -B-B- and R ^{3 are} defined in formula XX. Preferably, R ³ is hydrogen.

The conversion reaction is carried out by cyanidation, essentially as described above, to convert the 11α-hydroxy compound of formula VIII to the enamine of formula VII. Typically, the source of cyanide ions may be an alkali metal cyanide. The base is preferably pyrrolidine and / or tetramethylguanidine. A methanol solvent may also be used.

The products of formula XVB are new compounds that can be isolated by chromatography. These and other new compounds of formula XV serve as intermediates for the preparation of compounds of formula I, and especially of formula IA. The compounds of formula XV correspond to the structure:

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII. In the most preferred compounds of formula XV and formula XVB, -A-A- and -B-B- are —CH ₂ —CH ₂ - and R ³ is hydrogen.

In accordance with the hydrolysis described above to obtain diketone compounds of formula VI, enamines of formula XVB can be converted into diketones of formula XIVB;

where -A-A-, -B-B- and R ^{3 are} defined in formula XX. Preferably R ³ is hydrogen.

Particularly preferred for the synthesis of epoxymexrenone are those compounds of formula XIV, which are also encompassed by formula XIVB, as defined below.

The products of formula XIVB are new compounds that can be isolated by precipitation. These and other new compounds of formula XIV serve as intermediates for the preparation of compounds of formula I, and especially formula IA. The compounds of formula XIV correspond to the structure:

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII. In the most preferred compounds of formula XIV and XIVB, -A-A- and -B-B- are —CH ₂ —CH ₂ -, and R ³ is hydrogen.

Then the compounds of formula XIVB are converted to compounds of formula XXXI, mainly by the method described above, for the conversion of a diketone of formula VI to a hydroxy ester of formula V. In this case, intermediate XXXI must first be isolated:

and then carry out the reaction of its conversion into a product of the formula XXXII;

where -A-A-, -B-B- and R ^{3 are} defined in formula XX and R ^{1 is} defined in formula V. Preferably, R ³ is hydrogen. Preferred compounds of formula XXXI are compounds covered by formula IIA. Compounds of formula XXXI are converted to compounds of formula XXXII by the method described above or described in US Pat. No. 4,559,332.

A preferred compound of formula XIV is

4'S (4'α), 7'α-1 ', 2', 3 ', 4.4', 5.5 ', 6', 7 ', 8', 10 ', 12', 13 ', 14' , 15 ', 16'-hexadecahydro-10β-, 13'β-dimethyl-3', 5,20'-trioxospiro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopent [ a] phenanthrene] 5'-carbonitrile; and the compound of formula XV is 5'R (5'α), 7'β-20'-amino-1 ', 2', 3 ', - 4,5,6', 7 ', 8', 10 ', 12 ', 13', 14 ', 15', 16'-tetradecahydro-10'α, 13'α-dimethyl-3 ', 5-dioxospiro [furan-2 (3H), 17'α (5'H) - [ 7.4] metheno [4H] -cyclopenta [a] phenanthrene] -5'-carbonitrile. In a most preferred embodiment of the present invention, the complete reaction in Scheme 3 proceeds as follows:

Scheme 4.

The first three stages of Scheme 4 are similar to the stages of Scheme 3, i.e. a step for preparing an intermediate of formula XVII based on a compound of formula XX.

Then, the intermediate of formula XVII is epoxidized, for example, by the method described in US Pat. No. 4,559,332 to obtain a compound of formula XXIV:

where -A-A-, -B-B- and R ^{3 are} defined in formula XX. However, in a particularly preferred embodiment of the present invention, a substrate of formula XVII is epoxidized at 9.11-double bonds using an oxidizing agent containing an amide type peroxide activator, most preferably trichloroacetamide, in accordance with the method described above in Scheme 1 for converting the ester of formula II into the product of Formula I. The conditions and ratios of the reagents for this reaction are essentially the same as those described for the conversion of the ester of Formula II to epoxymexrenone. Particularly preferred compounds of formula XXIV are those compounds in which -A-A- and -B-B- are as defined for formula XIII and R ³ is hydrogen.

It has been found that the epoxidation reaction of a substrate of formula XVII can also give a very good yield using peracids, such as, for example, m-chloroperoxybenzoic acid. However, the trichloroacetamide reagent gives the best result with respect to minimizing the formation of a by-product from Bayer-Williger oxidation. This by-product can be removed, but this requires trituration from a solvent, such as ethyl acetate, followed by crystallization from another solvent, such as methylene chloride. The epoxy compound of formula XXIV is dehydrogenated to form a double bond between 6- and 7-carbons by reaction with a dehydrogenating agent (oxidizing agent) such as DDQ or chloranil, or using a sequential bromination / dehydrobromination reaction (or other halogenation / dehydrohalogenation reaction) with obtaining another new intermediate compound of formula XXIII.

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XX. Particularly preferred compounds of formula XXIII are those compounds wherein -A-A- and -B-B- are as defined for formula XIII, and R ³ is hydrogen.

Although direct oxidation is effective to obtain the product of formula XXIII, however, this reaction gives mainly low yields. Therefore, the oxidation reaction is preferably carried out in two stages, first, the substrate of formula XXIV is halogenated at the C-6 position, and then dehydrohalogenation is carried out to form a 6,7-olefin. The halogenation reaction is preferably carried out with an N-halogen-organic reagent, such as, for example, N-bromosuccinamide. The bromination reaction is carried out in a suitable solvent, such as, for example, acetonitrile, in the presence of a halogenation promoter, such as benzoyl peroxide. This reaction proceeds efficiently at temperatures ranging from about 50 to about 100 ° C, usually when heated to reflux, in a solvent such as carbon tetrachloride, acetonitrile, or mixtures thereof. However, it usually takes 4 to 10 hours to complete the reaction. After this, the reaction solvent is evaporated and the residue is dissolved in a water-immiscible solvent, for example ethyl acetate. The resulting solution is washed successively with a weak alkaline solution (such as alkali metal bicarbonate) and water, or preferably saturated brine, to minimize product losses, after which the solvent is evaporated and the residue is dissolved in another solvent (such as dimethylformamide) suitable for the dehydrohalogenation reaction.

A suitable dehydrohalogenation reagent, for example 1,4-diazabicyclo [2.2.2] octane (DABCO), is added to the solution together with an alkali metal halide such as LiBr, the solution is heated to a suitable reaction temperature, for example 60-80 ° C, and the reaction is carried out within a few hours, usually 4 to 15 hours, until dehydrobromination is complete. If necessary, an additional amount of dehydrobrominating reagent can be added during the reaction cycle to bring the reaction to completion. Then the product of formula XXIII can be isolated, for example, by adding water to precipitate the product, which is then separated by filtration and, preferably, washed with some more water. This product is preferably recrystallized, for example from dimethylformamide.

Products of formula XXIII, such as 9,11-epoxycanrenone, are novel compounds that can be isolated by extraction / crystallization. They are important as intermediates for the preparation of compounds of formula I, and especially of formula IA. For example, they can be used as substrates for the preparation of compounds of formula XXII.

Using basically the method described above for preparing compounds of formula VII, compounds of formula XXIII are reacted with a cyanide ion to produce new epoxyenamine compounds corresponding to formula XXII:

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XX. Particularly preferred compounds of formula XXII are those wherein -A-A- and -B-B- are as defined for formula XIII and R ³ is hydrogen.

The products of formula XXII are new compounds that can be isolated by precipitation and filtration. They are important as intermediates for the preparation of compounds of formula I, and especially of formula IA. In the most preferred compounds of formula XXII, -A-A- and -B-B- are —CH ₂ —CH ₂ -, and R ³ is hydrogen.

Using mainly the method described above for the preparation of compounds of formula VI, epoxy-amine compounds of formula XXII are converted into new epoxy-ketone compounds of formula XXI:

where -A-A-, -B-B-, R ³ , R ⁸ and R ^{9 are} defined in formula XIII. In the most preferred compounds of formula XXI, —A — A— and —B — B— are —CH ₂ —CH ₂ -, and R ³ is hydrogen.

The products of formula XXI are new compounds that can be isolated by precipitation and filtration. They are important as intermediates for the preparation of compounds of formula I, and especially of formula IA. Particularly preferred compounds of formula XXI are compounds in which -A-A- and -B-B- are as defined for formula XIII. In the most preferred compounds of formula XXI, —A — A— and —B — B— are —CH ₂ —CH ₂ -, and R ³ is hydrogen.

Using basically the method described above for preparing the hydroxy ester compounds of formula V from diketone compounds of formula VI, epoxy ketone compounds of formula XXI are converted to compounds of formula XXXII:

where -A-A-, -B-B- and R ^{3 are} defined in formula XX, and R ^{1 is} defined in formula V.

As in the reaction for converting a diketone of formula V to a hydroxy ester of formula VI, a 5-β-cyano-7-ester intermediate is also formed in the reaction for converting an epoxydiketone of formula XXI to compounds of formula XXXII. Both types of these 5-β-cyano-7-ester intermediates can be isolated by treating the corresponding diketone with an alcohol, such as methanol, in the presence of a base, such as triethylamine. Preferably, these intermediates are prepared by refluxing a mixture of diketone in alcohol, such as methanol, containing from about 0.1 to about 2 equivalents of triethylamine per mole of diketone, for about 4 to about 16 hours. The products are isolated in pure form by cooling the mixture to about 25 ° C, followed by filtration. The isolated intermediates can be converted to compounds of formula XXXII by treatment with a base, such as an alkali metal alkoxide, in a solvent, preferably an alcohol, such as methanol. The use of alkoxide in alcohol gives an equilibrium mixture similar to that which forms when the corresponding diketone of formula XXI is treated under the same conditions.

In addition, a 7β-ester of a compound of formula XXXII (e.g. 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene was found in the crude product of the final step of the method of Scheme 4 using chromatography -7β, 21-dicarboxylate, γ-lactone). The alkoxide and / or cyanide in solution react with each other with the conversion of the 7α-ester to an epimeric mixture of the 7α-ester and its 7β-ether epimer. Pure 7β-ester can be isolated from the epimeric mixture by selective crystallization.

Preferably, the compound of formula XXI is 4'S- (4'α), 7'α-9 ', 11α-epoxyhexadecahydro-10β-, 13'β-dimethyl-3', 5,20'-trioxospiro [furan-2 (3H), 17'β- [4,7] methano [17H] -cyclopenta [a] -phenanthrene-5'-carbonitrile; the compound of formula XXII is 5'R (5'α), 7'β-20'-amino-9,11β-epoxyhexadecahydro-10,13'-dimethyl-3 ', 5-dioxospiro [furan-2 (3H), 17 'α- (5'H) - [7,4] methene [4H] -cyclopenta [a] phenanthrene-5'-carbonitrile; and the compound of formula XXIII is 9,11α-epoxy-17α-hydroxy-3-oxopregna-4,6-diene-21-carboxylic acid, γ-lactone.

In a particularly preferred embodiment of the present invention, the complete reaction process of Scheme 4 is as follows:

Scheme 5.

The process of Scheme 5 is carried out on the basis of a substrate corresponding to the formula XXIX:

where: -A-A-, -B-B-, R ^{3 are} defined above in formula XX.

The following microorganisms are capable of 9α-hydroxylation of a compound of formula XXXV (such as androstenedione).

where: -A-A-, -B-B-, R ^{3 are} defined above in formula XIII, with the formation of the compounds of formula XXIX under conditions similar to those described in Example 19B:

Aspergillus niger ATCC 16888 and 26693, Corynespora cassiicola ATCC 16718, Curvularia clavata ATCC 22921, Mycobacterium fortuitum NRRL B8119, Nocardia canicruria ATCC 31548, Pycnosporium spp. ATCC 12231, Stysanus microsporus ATCC 2833, Syncephalastrum racemosum ATCC 18192 and Thamnostylum piriforme ATCC 8992.

The substrate corresponding to the formula XXIX, turn into a product of the formula XXVIII:

by reaction with trimethylorthoformate, where: -A-A-, -B-B- and R ^{3 are as} defined above in formula XX.

After the formation of compounds of formula XXVIII, these compounds are converted to compounds of formula XXVII by the method described above to convert a substrate of formula XX to a compound of formula XVII. The compounds of formula XXVII have the following structure:

where: -A-A-, -B-B-, R ^{3 are as} defined above in formula XX, and R ^x represents any of the standard hydroxy-protecting groups.

Alternatively, a C9-α-hydroxy group may be protected at an earlier stage of this synthesis scheme, if protection at this stage is desired, that is, a C9-hydroxy group of a compound of formula XXVIII or a C9-hydroxy group of a compound of formula XXIX can be protected by any from standard hydroxy protecting groups.

By the method described above to obtain compounds of formula XVI, compounds of formula XXVII are oxidized to give new compounds corresponding to formula XXVI

where: -A-A-, -B-B- and R ^{3 are} defined above in formula XX.

Particularly preferred compounds of formulas XXIX, XXVIII, XXVII and XXVI are compounds in which -A-A- and -B-B- are as defined for formula XIII and R ³ is hydrogen.

The products of formula XXVI are new compounds that can be isolated by precipitation / filtration. They can mainly be used as intermediates for the preparation of compounds of formula I, and especially compounds of formula IA. Particularly preferred compounds of formula XXVI are those wherein -A-A- and -B-B- are as defined for formula XIII and R ³ is hydrogen. In the most preferred compounds of formula XXVI, -A-A- and -B-B- are —CH ₂ —CH ₂ -, and R ³ is hydrogen.

By the method described above for cyanidation of compounds of formula VIII, the new intermediates of formula XXVI are converted to the new 9-hydroxyenamine intermediates of formula XXV

where: -A-A-, -B-B-, R ^{3 are} defined above in formula XX.

The products of formula XXV are novel compounds that can be isolated by precipitation / filtration. They are mainly used as intermediates for preparing compounds of formula I, and especially compounds of formula IA. Particularly preferred compounds of formula XXVI are those wherein -A-A- and -B-B- are as defined for formula XIII, and R ³ is hydrogen. In the most preferred compounds of formula XXVI, -A-A- and -B-B- are —CH ₂ —CH ₂ -, and R ³ is hydrogen.

By the method described above for the preparation of diketone compounds of formula VI, 9-hydroxyenamine intermediates of formula XXV are converted into diketone compounds of formula XIVB. It should be noted that in this case, this reaction is effective for the simultaneous hydrolysis of the enamine structure and dehydration at 9.11 positions for introducing a 9.11 double bond. The compound of formula XIV is then converted to the compound of formula XIII using the same step as described above in Scheme 3.

Preferably, the compound of formula XIV is 4'S (4'α), 7'α-1 ', 2', 3 ', 4.4', 5.5 ', 6', 7 ', 8', 10 ', 12' , 13 ', 14', 15 ', 16'-hexadecahydro-10β-13'β-dimethyl-3', 5,20'-trioxospiro [furan-2 (3H), 17'β- [4,7] methano [17H] -cyclopenta [a] phenanthrene] -5'-carbonitrile; the compound of formula XXV is 5'R (5'α), 7'β-20'-aminohexadec-hydro-9'β-hydroxy-10'α, 13'α-dimethyl-3 ', 5-dioxospiro [furan-2 (3H), 17'α (5'H) - [7,4] metheno [4H] -cyclopenta [a] -phenanthrene] -5'-carbonitrile; the compound of formula XXVI is 9α, 17α-dihydroxy-3-oxopregna-4,6-diene-21-carboxylic acid, γ-lactone; and the compound of formula XXVII is 9α, 17α-dihydroxy-3-oxo-pregn-4-en-21-carboxylic acid, γ-lactone.

In a particularly preferred embodiment of the invention, the entire method of Scheme 5 is as follows:

Scheme 6.

Scheme 6 represents an advantageous method for producing epoxymexrenone and other compounds corresponding to formula I, starting from 11α or 11β-hydroxylation of androstenedione or another compound of formula XXXV:

where: -A-A-, -B-B- and R ^{3 are as} defined above in formula XIII to give an intermediate corresponding to formula XXXVI or its corresponding 11β-hydroxy isomer

where: -A-A-, -B-B- and R ^{3 are as} defined above in formula XIII.

With the exception of the choice of substrate, this 11α-hydroxylation process is mainly carried out as described above for Scheme 1. The following microorganisms are capable of carrying out the 11α-hydroxylation of androstenedione or another compound of formula XXXV:

Absidia glauca ATCC 22752, Aspergillus flavipes ATCC 1030, Aspergillus foetidus ATCC 10254, Aspergillus fumigatus ATCC 26934, Aspergillus ochraceus NRRL 405 (ATCC 18500), Aspergillus niger ATCC 11394, Aspergus athumus 715 bomida oxysporum cepae ATCC 11171, Fusarium Lini ATCC IFO 7156, Gibberella fujikori ATCC 14842, Hypomyces chyrsospermus IMI 109891, Mycobacterium fortuitum NRRL B8119, Penicillum patulum ATCC 24550, Pycnosporium spp. ATCC 12231, Rhyzopus arrhizus ATCC 11145, Saccharopolyspora erythraea ATCC 11635, Thamnostylum piriforme ATCC 8992, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b, and Trichothecium roseum ATCC 825568CC ATCC 125685 ATCC 12568 ATCC.

The 11β-hydroxylation of androstenedione or another compound of formula XXXV is able to carry out the following microorganisms:

Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC 16888 and ATCC 26693, Epicoccum oryzae ATCC 7156, Curvularia lunata ATCC 12017, Cunninghamella blakesleeana ATCC 8688a and Pithomyces atro-olivaceous IFO 6651.

Then, 11α-hydroxyandrost-4-en-3,17-dione or another compound of formula XXXVI is converted into an 11α-hydroxy-3,4-enol ether of formula (101):

where: -A-A-, -B-B- and R ^{3 are} defined above in formula XIII, and R ¹¹ is methyl or other lower alkyl (C ₁ -C ₄ ) by reaction with an esterifying reagent such as trialkyl orthoformate, in the presence of an acid catalyst. To carry out this conversion, the 11α-hydroxysubstrate is acidified by mixing with an acid, such as, for example, benzenesulfonic acid hydrate or toluenesulfonic acid hydrate in a solvent, such as a lower alcohol, for example ethanol. Trialkyl orthoformate, preferably triethyl orthoformate, is introduced gradually over a period of 5-40 minutes, keeping the mixture cold, preferably at a temperature of from about 0 ° C to about 15 ° C. Then the mixture is heated and the reaction is carried out at a temperature of from 20 ° C to about 60 ° C. The reaction is preferably carried out at 30-50 ° C for 1-3 hours, and then heated under reflux for some time, usually for 2-6 hours, until the reaction is complete. The reaction mixture is cooled, preferably to 0 ° C.-15 ° C., and more preferably at about 5 ° C., and the solvent is removed in vacuo.

Using the same reaction scheme as described above in Scheme 3 to convert a compound of formula XX to a compound of formula XVII, the 17-spirolactone portion of formula XXXIII is introduced into a compound of formula 101. Thus, for example, a substrate of formula 101 can be reacted with ylide sulfonium in the presence of a base, such as an alkali metal hydroxide, in a suitable solvent, such as DMSO, to give an intermediate of formula 102:

where: -A-A-, R ³ , R ¹¹ and -B-B- are defined above in formula 101. An intermediate of formula 102 is then reacted with a malonic acid diester in the presence of an alkali metal alkoxide to give a five-membered spirolactone ring and an intermediate formula 103:

where: -A-A-, R ³ , R ¹¹ and -B-B- are defined above in formula 102, and R ¹² is C ₁ -C ₄ alkyl, preferably ethyl. Finally, the compound of formula 103 in a suitable solvent, such as dimethylformamide, is heated in the presence of an alkali metal halide, while splitting off, alkoxycarbonyl group and obtaining an intermediate compound of formula 104:

where again -A-A-, R ³ , R ¹¹ and -B-B- are defined above in formula 102.

The ether compound 3,4-enol 104 is then converted to a compound of formula XXIII, that is, a compound of formula VIII in which R ⁸ and R ⁹ taken together form a part of formula XXXIII. This oxidation step is carried out essentially in the same manner as the oxidation step was carried out to convert the compound of formula XXIV to the intermediate of formula XXIII in Synthesis Scheme 4. Direct oxidation can be carried out using a reagent such as 2,3-dichloro-5, 6-dicyano-1,4-benzoquinone (DDQ) or tetrachlorobenzoquinone (chloranil); or, preferably, two-stage oxidation is carried out first by bromination, for example using an N-halogen-brominating agent such as N-bromosuccinamide (NBS) or 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), and then by dehydrobromination with using a base, for example DABCO, in the presence of LiBr and at elevated temperature. If NBS is used for bromination, an acid must also be used to convert the 3-enol ester to enone. DBDMH, an ionic rather than free radical brominating reagent, is itself effective in the bromination and conversion of the enol ester to enon.

The compound of formula VIII is then converted to epoxymexrenone or another compound of formula I in the steps described above for Scheme 1.

Each of the intermediate compounds of formulas 101, 102, 103 and 104 is a new compound, which can be mainly used as an intermediate to produce epoxymextrenone or other compounds of formulas IA and I. In each of the compounds of formulas 101, 102, 103 and 104 -A-A- and -B-B- are —CH ₂ —CH ₂ -, and R ³ is hydrogen, lower alkyl, or lower alkoxy. R ^{3 is} preferably hydrogen. Most preferably, the compound of formula 101 is 3-ethoxy-11α-hydroxyandrost-3,5-dien-17-one; the compound of formula 102 is 3-ethoxy-spiro [androst-3,5-diene-17β, 2'-oxiran] -11α-ol, the compound of formula 103 is ethylhydro-3-ethoxy-11α-17α-dihydroxypregna-3,5-diene 21,21-dicarboxylate, gamma-lactone and a compound of formula 104 is 3-ethoxy-11α-17α-dihydroxy-pregna-3,5-diene-21-carboxylic acid, gamma-lactone.

In a particularly preferred embodiment, the entire process of Scheme 6 proceeds as follows:

It has been suggested that epoxymexrenone and other compounds of formula I can also be prepared from 11β-hydroxyandrostenedione or other compounds of formula XXXV that have been 11β-hydroxylated. In other words, epoxymexrenone and other compounds of formula I can be prepared by the general method described in Scheme 6 using either an α-hydroxylated substrate of formula XXXV or the corresponding β-hydroxylated substrate.

Scheme 7.

Scheme 7 represents the synthesis of epoxymexrenone and other compounds of formula I using an initial substrate containing β-sitosterol, cholesterol, stigmasterol or another compound of formula XXXVII:

where: -A-A-, R ³ and -B-B- are defined above in formula XIII; DD is —CH ₂ —CH ₂ - or —CH═CH—; and each of R ¹³ , R ¹⁴ , R ¹⁵ and R ^{16 is} independently selected from hydrogen or C ₁ -C ₄ alkyl.

R ^{3 is} preferably hydrogen.

In the first step in the synthesis of 11α-hydroxyandrostenedione or another compound of formula XXXVI, is obtained by biological conversion of a compound of formula XXXVII. The method of such biological prostration is carried out mainly by the method described above for 11α-hydroxylation of canrenone (or another substrate of formula XIII).

In the synthesis of 11α-hydroxyandrostenedione, 4-androsten-3,17-dione is first obtained by biological conversion of a compound of formula XXXVII. This initial biological conversion can be carried out by the method described in US patent No. 3759791, which in its entirety is introduced into the present description by reference. Then, 4-androsten-3,17-dione is converted to 11α-hydroxyandrostenedione, mainly by the method described above for 11α-hydroxylation of canrenone (or another substrate of formula XIII).

The rest of the synthesis of Scheme 7 is identical to Scheme 6. In a particularly preferred embodiment of the invention, the entire process of Scheme 7 proceeds as follows:

It has been suggested that epoxymexrenone and other compounds of formula I can also be prepared in accordance with the general method presented in Scheme 7, if the product of the biological conversion of β-sitosterol or other compounds of formula XXXVIII is 11β-hydroxyandrostenedione or other compounds of formula XXV that have been 11β-hydroxylated. In other words, epoxymexrenone and other compounds corresponding to formula I can be obtained by the general method presented in Scheme 7, if the biological conversion of β-sitosterol or other compounds of formula XXXVIII results in either an α-hydroxylated substrate of formula XXXV or the corresponding β-hydroxylated substrate .

Scheme 8.

A significant difficulty in the synthesis of epoxymexrenone and related compounds is the need for stereoselective administration of the α-alkoxycarbonyl substituent at 7-carbon, without causing undesirable modifications in other parts of the steroid structure. In accordance with the present invention, it was found that an efficient synthesis method for introducing a 7α-alkoxycarbonyl substituent includes the following steps: (i) initial cyanidation in the 7-carbon steroid, (ii) hydrolysis of the 7-cyanosteroid to form a mixture of 7α-carboxylic acid and 7β- carboxylic acid steroids, (iii) the formation of a 5,7-lactone steroid from a 7α-carboxylic acid steroid, and (iv) the separation of a 7β-carboxylic acid steroid from a 5,7-lactone steroid. As a result of the ring-mediated ring opening reaction that occurs between the 5,7-lactone steroid and the alkylating reagent, the desired 7α-alkoxycarbonyl steroid is obtained.

In accordance with this, the process of Scheme 8 is usually aimed at obtaining a 3-keto-7α-alkoxycarbonyl-substituted Δ ^4,5 -steroid, involving the reaction of an alkylating reagent with 3-keto-4,5-dihydro-5,7-lactone steroid substrate in the presence of a base. The lactone substrate is substituted by a keto group at the 3-position of carbon and, in addition, contains a part:

where C (5) represents 5-carbon, and C (7) represents 7-carbon of the steroid structure of the substrate. The conversion of 5,7-lactone to 7α-alkoxycarbonyl is preferably carried out by reaction with an alkyl halide in the presence of a base. The alkyl halide reagent is preferably iodide, and most preferably methyl iodide.

Furthermore, in accordance with the present invention, an advantageous process has been developed for the preparation of the 4,5-dihydro-5,7-lactone steroid compound described above. In this method, the 3-keto-Δ ^4,5 -7α-cyano-substituted steroid substrate is converted to 7-carboxylic acid, and this acid, in turn, is reacted with trialakylorthoformate in an acidified solvent, lower alcohol, to give 5.7 lactone. The reaction with orthoformate esters promotes the conversion of the 3-keto group to a 3-acyclic or cyclic ketal-5,7-lactone (it should be noted that lactone is formed first). A preferred 3-ketal-5,7-lactone is 3-dialkyl-ketal-5,7-lactone. More preferably, the alkyl part of the alcohol solvent is the same as the alkyl part of the alkoxy groups of the orthoformate (and most preferably all are methyl), because: the alkoxy parts of the ketal can be derived from orthoformate or from alcohol; mixed ketals are not preferred; and 3-dimethoxy is preferred. If the ketal is ethylene ketal, then the alkyl portion of the alcohol solvent need not be the same as the alkyl portion of the alkoxy groups of the orthoformate. 3-ketal-5,7-lactone is readily hydrolyzed to form 3-keto-5,7-lactone, a crystalline compound that can be easily purified. Since only 7α-carboxylic acid undergoes a lactonization reaction, complete stereospecificity is ensured. The 7β acid can then be removed from the reaction mixture as a salt, for example by treating the 7β acid with a weak base such as sodium bicarbonate.

7-cyano-substrate for the production of 5,7-lactone can be obtained in a known manner. For example, a substrate unsubstituted at the 7-position of carbon can be reacted with a slight excess of cyanide ion, preferably from about 1.05 to about 1.25 equivalents, per equivalent of substrate in a slightly acidified solution containing a water / DMSO solvent mixture. Preferably, the reaction mixture comprises carboxylic acid, for example about one equivalent of acetic acid per equivalent of substrate. In this case, both the 7α- and 7β-CN isomers are formed, the 7α-isomer being the main isomer. The 7α-cyano steroid can be isolated in a standard manner. Other methods known to those skilled in the art can be used in this additional preparation.

Basically, in accordance with Scheme 8, 5,7-lactone can be formed from an intermediate 7-carboxy compound (which itself is obtained by hydrolysis of an intermediate 7-cyano compound), which is substituted at the 17-position or a keto group, either R ⁸ or R ⁹ , where R ⁸ and R ^{9 are} defined above, and which has an aliphatic, olefinic, epoxy or hydroxy-substituted configuration in C-9 and C-11, i.e.

where -A-A-, -B-B- and R ^{3 are as} defined above, R ⁸⁰ and R ⁹⁰ are the same as R ⁸ and R ⁹ , or R ⁸⁰ and R ⁹⁰ taken together form a keto, R ^{18 is} defined below for Scheme 9, a -E-E- is selected from

The compound of formula XLII is then converted to 7α-alkoxy-carbonyl;

In each of the compounds XL, XLI, XLII and XLVIII, R ⁸⁰ and R ⁹⁰ , taken together, are preferably keto or

where Y ¹ , Y ² , X and C (17) are defined above, and most preferably, if R ⁸⁰ and R ⁹⁰ taken together are

R ^{3 is} preferably H, R ^{1 is} preferably methoxycarbonyl, and —A — A— and —B — B— are preferably —CH ₂ —CH ₂ -. It should be noted that these reactions can also be carried out with a 3-keto group, protected by its transformation into each ether or ketal form and maintaining it in this form throughout the sequence of reactions. Alternative methods of Scheme 8 involve the use of various intermediates encompassed by formulas XLI and XLII as defined above.

It should be noted that the reagent used to form 5.7-lactone from 3-keto-Δ ^4,5 -7-carboxylic acid in Scheme 8 is trialkyl orthoformate, the same reagent used to convert 11α-hydroxyandrostenedione to intermediate 3 enol-ether-3,5-diene-11α-hydroxy-compound 101 of Scheme 6, while it is obvious that the path along which Reaction Scheme 8 is carried out depends on substitution at C-7. As a result of the reaction with orthoformate in the presence of H ⁺ , an intermediate carbonium ion is formed having a carboxyl in position C-7 and a positive charge at equilibrium between C-3 and C-5. After the loss of the proton, the C-3 carbonyl ion gives a compound of formula 101, and the C-5 carbonium ion gives a lactone. In the case of hydrogen at C-7, it is obvious that mainly 3,5-diene-3-alkoxy (enol ether) is formed due to conjugation of double bonds. In the case of the 7α-CO ₂ substituent at C-7, the C-5 carbonyl ion is captured by carboxy and a 5.7-lactone is formed. At this stage, the 3-keto group is predominantly converted to ketal, which contributes to the completion of the reaction.

Preferred options for Scheme 8 are described below in Schemes 9 and 10.

Scheme 9.

Scheme 9 begins with the use of the same substrate that was used in Scheme 4, that is, the compounds of formula XX. This substrate is first oxidized to a compound of formula B;

where: -A-A-, R ³ and -B-B- are defined above in formula XIII.

The oxidation reaction is carried out in accordance with any of the reaction schemes described above to convert a compound of formula XXIV to an intermediate of formula XXIII in the synthesis of Scheme 4. Using the methods described for scheme 8, a compound of formula B is converted to a 7-cyano intermediate of formula C :

where -A-A-, R ³ and -B-B- are defined in formula XIII. The compound of formula C is then converted to a 5.7-lactone of formula D:

where -A-A-, R ³ and -B-B- are defined in formula XIII, and R ¹⁷ is C ₁ -C ₄ alkyl, using the trialkyl orthoformate reagent used previously in Scheme 6. 5,7-Lactone of formula D is easily isolated from unreacted compound 7-β-COOH, for example, by removing the acid by washing with bicarbonate, and thereby creating the desired stereochemical C-7 structure and preventing epimerization in subsequent reactions, which are carried out under basic conditions. The esterification of lactone by reaction with an alkyl halide, as described in Scheme 8, gives an ester intermediate of formula II.

Continuing the synthesis of Scheme 9, the compound of formula D is converted to the compound of formula II. Using a 3-keto group protected by conversion to a ketal, the 20-spiroxane group of formula XXXIII is selectively introduced into the 17-position according to the reaction scheme described above for Schemes 3 and 6 (see above), and a compound of formula E is obtained :

Since the 3-ketone is protected, the hydrolysis conditions can be selected so that they are optimal for exposure to the 17-ketone without the formation of by-products through the reaction in the 3-position. After hydrolysis of the 3-ketal compound of formula E into a structure with a 3-keto group of formula F

the latter intermediate is reacted with an alkyl iodide in the presence of a base according to Transformation Scheme 8 to give an intermediate ester of formula II. Finally, the latter intermediate is converted to epoxymexrenone or another compound of formula I using any of the methods described above for Scheme 1.

Scheme 9 has advantages not only because of the ability to control the stereochemistry imparted by the 5.7-lactone intermediate, but it also provides additional advantages in that it is possible to use the hydrolysis conditions over a wide range without affecting 17-spirolactone.

Like the reactions of other synthesis schemes of the present invention, the reactions of Scheme 9 can be used to convert substrates other than those specifically described above. So, for example, the conversion of 3-keto or 3-ketal-7-cyano steroids to 3-keto or 3-ketal-5,7-lactone, or the conversion of 3-keto or 3-ketal-5,7- the lactone to 7α-alkoxycarbonyl can be carried out using compounds substituted on the 17-carbon with the groups R ⁸ and R ⁹ defined above, or, more preferably, a substituent of the formula:

where X, Y ¹ and Y ^{2 are} defined above, and C (17) means 17-carbon. However, these important advantages are realized, especially from the point of view of the efficiency of the method, by carrying out reactions in a certain sequence using 17-keto-substrates and in accordance with the specific reaction scheme described above for the introduction of 17-spirolactone and 7α-alkoxycarbonyl in 3-keto Δ ^{9.11 is a} steroid.

The lactones of formulas D, E and F are new compounds that can be used to produce epoxymexrenone and other compounds of formulas I and IA in accordance with Synthesis Scheme 9. In these compounds, -A-A- and -B-B- are preferably -CH ₂ —CH ₂ -, and R ³ represents hydrogen, lower alkyl or lower alkoxy. Most preferred is a compound of formula D in which R ¹⁷ is methoxy.

In a particularly preferred embodiment of the invention, the entire process of Scheme 9 proceeds as follows:

Scheme 10.

Scheme 10 is the same as Scheme 9 up to the formation of the intermediate 7-cyano compound of formula C. In the next step of Scheme 10, the 7-cyano steroid is reacted with a trialkyl orthoformate in an alkanol solvent, preferably with trimethyl orthoformate in methanol, while protecting 3- keto and 17-keto groups by converting the first compound into an enol ether, and the latter into a ketal. The 7-cyano group is then reduced to 7-formyl, for example by reaction with dialkylaluminum hydride, preferably with diisobutylaluminum hydride, whereby a compound of formula 203 is obtained:

where -A-A-, R ³ and -B-B- are defined above in formula XIII, and R ¹⁸ is C ₁ -C ₄ alkyl.

Before protecting the keto groups, as described above, their reduction is prevented using dialkyl aluminum hydride. The intermediate of formula 203 is then reacted with dilute aqueous acid to selectively hydrolyze 17-ketal in the presence of an excess of alcohol (R ¹⁹ OH), whereby an intermediate of formula 204 is obtained:

where R ^{19 is} selected from lower alkyl (preferably C ₁ -C ₄ ), or the R ¹⁹ groups at the 3-position form a cyclic O, O-hydroxyalkyleneoxy substituent at 3-carbon. Semi-acetal [204] is further protected by treatment with alkanol (R ¹⁹ OH) in the presence of anhydrous acid to give an intermediate of formula 205:

where: -A-A-, -B-B-, R ³ and R ^{19 are as} defined above, and R ²⁰ is C ₁ -C ₄ alkyl.

The 17-Spirolactone moiety can then be introduced in accordance with the reaction steps described above for Schemes 3 and 6, which are carried out in the following sequence:

where -A-A-, -B-B-, R ³ , R ¹⁹ and R ^{20 are as} defined above, and R ²⁵ is C ₁ -C ₄ alkyl.

After that, the 3-position is deprotected by the standard hydrolysis reaction with the repeated introduction of the 3-keto group and 5,7-hemiacetal and get another intermediate compound corresponding to the formula 209:

where -A-A-, -B-B- and R ^{3 are} defined above.

Then, the 9.11-epoxy moiety is introduced by any of the methods described above to convert the compounds of formula II to compounds of the formula I. Under the oxidizing conditions of the epoxidation reaction, the half-acetal is partially converted to 5.7-lactone, resulting in the production of another intermediate compound corresponding to formula 211 :

where: -A-A-, -B-B- and R ^{3 are} defined above.

Any remaining reaction product of the intermediate 9,11-epoxy-5,7-semi-acetal of formula 210:

where: -A-A-, -B-B- and R ^{3 are} defined above, readily oxidized by standard methods to give the compound of formula 211. And finally, the intermediate of formula 211 is converted to epoxymexrenone or another compound of formula I by the method described in Scheme 8 to convert 5,7-lactone to a 7α-alkoxy carbonyl compound. Thus, the entire Scheme 10 proceeds as illustrated below; but it should be noted that at least the following steps can be carried out in situ without isolation of the intermediate. In general, the synthesis of Scheme 10 is as follows:

As in the case of Scheme 9, the reactions described above for Scheme 10 have important advantages, especially from the point of view of the efficiency of the method; and at the same time, the new reactions of Scheme 10 also have a more general application with respect to substrates that are not substrates specifically described above. For example, the introduction of a 7-formyl group into the ether of a 3-enol steroid, protection of the obtained 7-formyl-Δ-5,6-3,4-enol ether, hydrolysis with the formation of 5,7-hemi-acetal, and subsequent release can be carried out steroids substituted at the 17-position with R ⁸ and R ⁹ groups as defined above or, more specifically, with a substituent of the formula:

where each of X, Y ¹ , Y ² and C (17) are defined above.

Alternative methods of Scheme 10 involve the use of various intermediates encompassed by Formulas A203-A210, respectively, described above. Each of the intermediates of formula A203-A211 is a new compound that can be used to prepare epoxymexrenone and other compounds of formula I and IA in accordance with Synthesis Scheme 10.

In a particularly preferred embodiment of the invention, the entire process of Scheme 10 proceeds as follows:

Based on the several schemes illustrated above, it should be noted that the reaction stages selected for use in the methods of the present invention provide significant adaptability in the production of epoxymexrenone and related compounds. The key distinguishing features of these methods are, inter alia: (a) the biological conversion of a substrate such as canrenone, androstenedione or β-sitosterol to an 11α or 9α-hydroxy derivative (with the simultaneous conversion of β-sitosterol to a 17-keto structure; ( b) introducing a 9,11 double bond by dehydrating a compound containing either an 11α- or 9α-hydroxy group, followed by introducing an epoxy group by oxidizing the 9,11 double bond; (c) attaching 7α-alkoxycarbonyl through the formation of enamine hydrolysis of enamine to form a wild the tones and reactions of this diketone with an alkali metal alkoxide; (d) the formation of a 20-spiroxane ring at the 17-position; (e) the formation of 5,7-lactone and the esterification of this lactone with the formation of 7-alkoxycarbonyl; (f) protection of 3-ketone by conversion of 3-enol or 3-ketal into the ether during a series of transformations in other positions (including the formation of a 20-spiroxane ring in the 17-position) With some limitations, these stages of the four-component process (b) - (d) can be carried out almost in any order. Process steps (e) and (f) have comparative adaptability. They provide a way to obtain epoxymexrenone and other compounds of formula I and have been greatly simplified compared to the method described in US Pat. No. 4,559,332. In addition, they provide important advantages in terms of productivity and yield.

In the descriptions of the reaction schemes presented above, the preparation, isolation and purification of the reaction products can mainly be carried out by methods well known in the art. Unless otherwise indicated, conditions, solvents and reagents are either standard, or they are not critical, or both. However, some of the specific procedures specifically described above have advantages that provide an increase in the overall yield and / or productivity of the various process steps and schemes of the process, and / or the high quality of the intermediate and final 9,11-epoxy steroid products.

The effective use of the 20-spiroxane compounds produced in accordance with the present invention is described in US Pat. No. 4,559,332 (Grob), which is incorporated herein by reference in its entirety.

The 20-Spiroxane compounds obtained in accordance with the present invention are distinguished by their good biological properties and are therefore valuable pharmaceutically active ingredients. For example, they have a strong aldosterone antagonistic effect, which consists in the fact that they reduce and normalize the excessively high accumulation of sodium and excretion of potassium caused by aldosterone. Therefore, they act as potassium-retaining diuretics and can have important therapeutic applications, for example, for the treatment of hypertension, heart failure, or cirrhosis.

20-spiroxane derivatives having aldosterone antagonistic effects are known, see, for example, Fieser & Fieser: Steroids; p.708 (Reinhold Publ.Corp., New York, 1959) and the description of British patent No. 1041534; 17β-hydroxy-21-carboxylic acids and their salts with similar activity are also known, see, for example, US Pat. No. 3,849,404. However, compounds of this type, used to date as medicines, have significant drawbacks in that they always have some secular-specific activity, which sooner or later causes adverse effects with conventional long-term therapy. Adverse effects that can be attributed to the anti-androgenic activity of known anti-aldosterone preparations are particularly undesirable.

The methods, processes and compositions of the present invention, as well as the conditions and reagents used in the present invention, are further described in the following examples.

EXAMPLE 1

Mowed agar cultures were obtained using the culture medium described in Table 1

Table 1. YPDA (medium for mowed agar crops and cups) yeast extract 20 g peptone 20 g glucose 20 g agar 20 g distilled water quantity
- pH 6.7
- bringing to pH 5 by adding 10% wt./about N ₃ PO ₄ up to 1000 ml Distribution:
- for slanted media:
7.5 ml in test tubes 180 × 18 mm
- for cups (10 cm ⌀)
25 ml in test tubes 200 × 20 mm
- sterilization at 120 ° C for 20 minutes
- pH after sterilization: 5

To produce cultures of the first generation, Aspergillus ochraceus colonies were suspended in distilled water (2 ml) in a test tube; and 0.15 ml aliquots of this suspension were applied to each of the beveled media, which were obtained as described above. These beveled media were incubated for seven days at 25 ° C, after which a surface culture appeared, which was a white fluffy mycelium. The reverse side was painted orange in its lower part, and in the upper part - yellow-orange.

The first generation mowed cultures were suspended in a sterile solution (4 ml) containing a Tween 80 nonionic surfactant (3% by weight), and 0.15 ml aliquots of this suspension were used to inoculate the second generation mowed culture, which was obtained using medium for cultivation described in Table 2.

Table 2. Second generation crop media and standard beveled media malt extract 20 g Peptone 1 g Glucose 20 g Agar 20 g distilled water, available
- pH 5.3
- distribution in 1.5 ml tubes (180 × 18 mm)
- sterilization at 120 ° C for 20 minutes up to 1000 ml

The second generation mowed cultures were incubated for 10 days at 25 ° C, resulting in a heavy mass of golden-colored spores; the reverse side was painted brown-orange.

A protective medium was obtained having the composition shown in Table 3.

Table 3. Protective environment Separated milk 10 g Distilled water 100 ml Separated milk was added to a 250 ml flask containing 100 ml of distilled water at 50 ° C. Sterilized at 120 ° C for 15 minutes. Cooled at 33 ° C and used until the end of the day.

Cultures from five second generation crop beveled cultures were suspended in a protective solution (15 ml) in a 100 ml flask. The suspension was divided into aliquots (0.5 ml each) in 100 × 10 mm lyophilization tubes. These cultures were pre-frozen at a temperature of from -70 ° C to -80 ° C in an acetone / dry ice bath for 20 minutes, and then immediately transferred to a dried chamber, pre-cooled to a temperature of from -40 ° C to -50 ° C . Pre-chilled aliquots were lyophilized at a residual pressure of 50 μm Hg. and at a temperature of ≤ -30 ° C. At the end of lyophilization, two or three granules of sterile silica gel were added to each tube with a moisture indicator and a hermetically sealed flame.

To obtain a uterine beveled culture suitable for large-scale fermentation, one aliquot of the lyophilized culture, which was obtained by the method described above, was suspended in distilled water (1 ml) and 0.15 ml aliquots of this suspension were used to inoculate the beveled cultures obtained with using a culture medium having the composition shown in Table 2. These uterine beveled cultures were incubated for seven days at 25 ° C. At the end of incubation, the culture grown on mowed agar was stored at 4 ° C.

To obtain a standard beveled culture, the culture obtained from the mother culture on beveled agar was suspended in a sterile solution (4 ml) containing Tween 80 (3% by weight), and the resulting suspension was distributed in 0.15 ml aliquots to the beveled cultures, which were coated with the culture medium described in Table 2. Standard beveled cultures can be used to inoculate primary seed flasks for laboratory or industrial fermentation.

To prepare the primary culture in the seed flask, the culture from the standard beveled culture, which was obtained as described above, was removed and suspended in a solution (10 ml) containing Tween 80 (3% wt.). A 0.1-aliquot of the resulting suspension was introduced into a 500 ml septum flask containing a culture medium having the composition shown in Table 4.

Table 4. (for primary culture and for transformation in the flask and in the round bottom flask) Glucose 20 g Peptone 20 g yeast autolysate 20 g distilled water, available
- pH 5.2
- bringing to 5.8 by adding 20% NaOH
- distribution in a 500 ml flask with a septum, 100 ml
- distribution in round-bottom 2000 ml flasks with partitions (3 partitions), 500 ml
- sterilization at 120 ° C for 20 minutes
- pH after sterilization of about 5.7

The seed flask was incubated on a rotating shaker (200 rpm, offset 5 cm) for 24 hours at 28 ° C, whereby a culture was obtained in the form of a granule-like mycelium with granules having diameters of 3-4 mm. After evaluation under a microscope, it was found that the seed culture is a pure crop, has synematic growth and large hyphae, well twisted into a spiral. The pH of the suspension was 5.4-5.6. PMV was 5-8%, as determined by centrifugation (3000 rpm × 5 min).

A culture for transformation in a flask was obtained by inoculating a culture medium (100 ml) having the composition shown in Table 4 in a second 500 ml shaker flask containing biomass (1 ml) from a seed culture flask. The resulting mixture was incubated on a rotating shaker (200 rpm, offset 5 cm) for 18 hours at 28 ° C. After examining the culture, it was found that it contains granule-like mycelium with granules with a diameter of 3-4 mm. After evaluation under a microscope, it was found that the seed culture is a pure culture, exhibits synematic and filamentous growth, in which the apical (apical) cells consisted entirely of cytoplasm, and older cells were slightly vacuolated. The pH of the culture suspension was 5-5.2, and PMV was determined by centrifugation to 10% -15%. Accordingly, the culture was found to be suitable for the conversion of canrenone to 11α-hydroxycanrenone.

Canrenone (1 g) was finely ground to form particles of about 5 microns in size and suspended in sterile water (20 ml). To this suspension was added: 40% (w / v) sterile glucose solution; 16% (w / v) sterile autolized yeast solution and sterile antibiotic solution; the contents of all ingredients are indicated for 0 hours of reaction time in Table 5. An antibiotic solution was prepared by dissolving kanamycin sulfate (40 mg), tetracycline · HCl (40 mg) and cephalexin (200 mg) in water (100 ml). A steroid suspension, glucose solution and autolysed yeast solution were gradually added to the culture contained in the shaker flask.

Table 5. Characteristic additions of steroid and solutions (additives and antibiotics) during the biological conversion of canrenone in a shaker flask Reaction time, hours Steroid suspension Glucose solution, ml Autolized yeast, ml The solution of antibiotics, ml ml approx. mg 0 1 fifty 1 0.5 1 8 2 100 2 1 24 2 100 1 0.5 1 32 5 250 2 1 48 2 100 1 0.5 1 56 5 250 2 1 72 3 150 1 0.5 1 90

As the reaction progressed, the reaction mixture was periodically analyzed to determine the glucose content and the degree of conversion to 11α-hydroxycanrenone was determined by thin-layer chromatography. During the reaction, an additional amount of canrenone substrate was added to the mixture for the fermentation reaction in controlled amounts to maintain a glucose level of about 0.1% by weight. The addition scheme for a steroid suspension of glucose solution, autolysed yeast solution and antibiotic solution is presented in Table 5. The transformation reaction was carried out for 96 hours at 25 ° C on a rotary shaker (200 rpm and a displacement of 5 cm). During fermentation, the pH was in the range of 4.5-6. When PMV rose to 60% or higher, 10 ml of a portion of the broth culture was removed and replaced with 10 ml of distilled water. The disappearance of canrenone and the appearance of 11α-hydroxycanrenone were continuously monitored during the course of the reaction by taking samples at time intervals 4, 7, 23, 31, 47, 55, 71, 80, and 96 hours after the start of the fermentation cycle and subsequent analysis of these samples using TLC . The progress of the reaction, determined from these samples, is shown in Table 6.

Table 6. The passage of the biological conversion reaction of canrenone in a shaker flask Time watch Degree of transformation Canrenon Rf.
RF. = 0.81 11α-hydroxycanrenone
RF. = 0.29 0 100 0,0 4 fifty fifty 7 twenty 80 23 twenty 80 31 thirty 70 47 twenty 80 55 thirty 70 71 25 75 80 fifteen 85 96 ~ 10 ~ 90

Example 2

The primary culture in the seed flask was obtained by the method described in Example 1. Received a nutrient mixture having the composition shown in Table 7

Table 7. Culture for transformation in a 10 liter glass fermenter quantity g / l Glucose 80 g twenty Peptone 80 g twenty autolysed yeast 80 g twenty defoamer SAG 471 0.5 g deionized water ext. up to 4 l - sterilization of the empty fermenter for 30 min at 130 ° C - loading 3 liters of deionized water, heating at 40 ° C - adding while stirring the components of the medium - stirring for 15 minutes, bringing to a volume of 3.9 l - pH 5.1 - bringing to 5.8 using 20% wt./about. NaOH, - sterilization at 120 ° C × 20 minutes - pH after sterilization = 5.5-5.7

The initial loading of this nutrient mixture (4 L) was introduced into the fermenter for transformation, having a geometric volume of 10 L. This fermenter had a cylindrical configuration and a height to diameter ratio of 2.58. It was equipped with a turbine mixer (400 rpm) having two No. 2 disc wheels with 6 blades each. The outer diameter of these mixer impellers was 80 mm, each blade had a radial size of 25 mm and a height of 300 mm, with the upper wheel located 280 mm lower from the top of the vessel, the lower wheel located 365 mm lower from the top of the vessel, and the partition for the vessel had a height of 210 mm and were located radially in the inner part at a distance of 25 mm from the inner vertical wall of the vessel.

A seed culture (40 ml) was mixed with a nutrient load in a fermenter and a culture for transformation was obtained by incubation for 22 hours at 28 ° C, at an aeration rate of 0.5 l / l / min and at a pressure of 0.5 kg / cm ² . After 22 hours, PMV culture was 20-25%, and pH = 5-5.2.

A suspension was prepared containing canrenone (80 g) in sterile water (400 ml), and a 10 ml portion was added to the mixture in the fermenter for transformation. At the same time, 40% (w / v) sterile glucose solution, 16% (w / v) sterilized autolysed yeast solution and a sterile antibiotic solution were added in the ratios indicated in Table 8 for a reaction time of 0 hours. An antibiotic solution was prepared by the method described in Example 1.

Table 8. Characteristic additions of steroid and solutions (additives and antibiotics) during the biological conversion of canrenone in a 10 liter glass fermenter Reaction time, hours Steroid suspension Glucose solution, ml Autolized yeast, ml The solution of antibiotics, ml ml approx. g 0 10 4 25 12.5 40 4 25 12.5 8 10 4 25 12.5 12 25 12.5 16 10 4 25 12.5 twenty 25 12.5 24 10 4 25 12.5 40 28 10 4 25 12.5 32 12.5 5 25 12.5 36 12.5 5 25 12.5 40 12.5 5 25 12.5 44 12.5 5 25 12.5 48 12.5 5 25 12.5 40 52 12.5 5 25 12.5 56 12.5 5 25 12.5 60 12.5 5 25 12.5 64 12.5 5 25 12.5 68 12.5 5 25 12.5 72 12.5 5 25 12.5 40 76 12.5 5 25 12.5 80 84 88

As the reaction progressed, the reaction mixture was periodically analyzed to determine the glucose content and the degree of conversion to 11α-hydroxycanrenone was determined by thin-layer chromatography. Based on TLC analysis of the reaction broth samples carried out as described below, additional canrenone was added to the reaction mixture as the canrenone substrate was consumed. Glucose levels were also monitored and when the glucose concentration dropped to about 0.05% of the mass. or lower, an additional amount of glucose solution was added to bring the concentration to about 0.25% of the mass. During the reaction cycle, nutrients and antibiotics were also added at various time intervals. The scheme for adding a steroid suspension, glucose solution, autolysed yeast solution and antibiotic solution is presented in Table 8. The transformation reaction lasted 90 hours at an aeration rate of 0.5 air volume per liquid volume for 1 minute (r / r / min) at a positive pressure in the head parts 0.3 kg / cm ² . The temperature was maintained at 28 ° C until the PMV reached 45%, after which the temperature was reduced to 26 ° C and this temperature was maintained as the PMV increased from 45% to 60%, and then it was maintained at 24 ° C. The initial stirring speed was 400 rpm, and after 40 hours it was increased to 700 rpm. The pH was maintained between 4.7-5.3 by adding 2M phosphoric acid or 2M NaOH, as indicated. As the foam formed, it was quenched by adding a few drops of SAG 471 antifoam. During the reaction, the disappearance of canrenone and the appearance of 11α-hydroxycanrenone were monitored at 4-hour intervals using TLC analysis of the broth samples. When the largest amount of canrenone disappeared from the broth, additional amounts of canrenone were added.

After adding the total amount of canrenone, the reaction was completed when TLC analysis showed that the concentration of the canrenone substrate with respect to the 11α-hydroxycanrenone product was reduced to about 5%.

At the end of the reaction cycle, the fermentation broth was filtered through gauze to separate the mycelium from the liquid broth. The mycelial fraction was resuspended in ethyl acetate using about 65 volumes (5.2 liters) per gram of canrenone loaded during the reaction. A suspension of mycelium in ethyl acetate was heated under reflux for one hour with stirring, and then cooled to about 20 ° C and filtered on a Buchner funnel. The mycelial precipitate was washed successively with ethyl acetate (5 vol. Per one gram of canrenone charge; 0.4 L) and deionized water (500 ml) to remove the ethyl acetate extract from the precipitate. The filter cake was discarded. The enriched extract, solvent washing and water washing were collected in a separator and then left to stand for 2 hours to separate the phases.

Then the aqueous phase was discarded, and the organic phase was concentrated in vacuo to obtain a residual volume of 350 ml. The bottom residue was cooled to 15 ° C and this temperature was maintained with stirring for about one hour. The resulting suspension was filtered to remove the crystalline product, and the filter cake was washed with ethyl acetate (40 ml). After drying, the yield of 11α-hydroxycanrenone was determined, which was 60 g.

Example 3

A spore suspension was obtained from a standard beveled culture by the method described in Example 1. In a 2000 ml round bottom flask with septa (3 septa, each 50 mm × 30 mm in size) containing a nutrient solution (500 ml) having the composition shown in Table 4 , an aliquot (0.5 ml) of the spore suspension was introduced. The resulting mixture was incubated in a flask for 24 hours at 25 ° C on a batch shaker (120 shakes per min; offset 5 cm), resulting in a culture which, as was established after observation under a microscope, was a pure culture with gifs, well-twisted into a spiral. The pH of the culture was about 5.3-5.5, and PMV (as determined by centrifugation at 3000 rpm for 5 min) was 8-10%.

Using the culture thus obtained, the seed culture was obtained in a stainless steel fermenter having a vertical cylindrical configuration, a geometric volume of 160 L and a relative format of 2.31 (height = 985 mm, diameter = 425 mm). This fermenter was equipped with a turbine mixer (400 rpm) having two disk impellers, each of which had 6 blades with an outer diameter of 240 mm, and each blade had a radial size of 80 mm and a height of 50 mm. The upper impeller was located at a depth of 780 mm from the top of the fermenter, and the second impeller was located at a depth of 995 mm from the top of the fermenter. The vertical partitions had a height of 890 mm and were located radially inside at a distance of 40 mm from the inner vertical wall of the fermenter. The mixer worked at a speed of 170 rpm. First, a nutrient mixture (100 L) having the composition shown in Table 9 was introduced into the fermenter, and then a portion of the pre-inoculum (1 L) prepared as described above and having a pH of 5.7 was introduced.

Table 9. For a vegetative culture in a 160 liter fermenter, about 8 liters needed for a seed production fermenter quantity g / l Glucose 2 kg twenty Peptone 2 kg twenty autolysed yeast 2 kg twenty defoamer SAG 471 0.010 kg trace amount deionized water ext. up to 100 l - sterilization of an empty fermenter for 1 hour at 130 ° C - load 6 l of deionized water; heating at 40 ° C - adding, stirring, components of the medium; - stirring for 15 minutes, - bringing to a volume of 95 l - sterilization at 121 ° C, 30 minutes - pH after sterilization 5.7 - adding sterile deionized water up to 100 l

The inoculated mixture was incubated for 22 hours at an aeration rate of 0.5 l / l / min and at a head pressure of 0.5 kg / cm ² . The temperature was maintained at 28 ° C until the PMV reached 25%, after which the temperature was reduced to 25 ° C. The pH was maintained in the range of 5.1-5.3. The increase in mycelium volume is shown in Table 10, which also shows the pH and dissolved oxygen profiles in the seed culture reaction.

Table 10. The growth time of mycelium in the reaction of fermentation of a seed crop Fermentation period, hours pH The volume of precipitated mycelium (PMV),% (3000 rpm / 5 min) Dissolved oxygen,% 0 5.7 ± 0.1 100 4 5.7 ± 0.1 100 8 5.7 ± 0.1 12 ± 3 85 ± 5 12 5.7 ± 0.1 15 ± 3 72 ± 5 16 5.5 ± 0.1 25 ± 5 40 ± 5 twenty 5.4 ± 0.1 30 ± 5 35 ± 5 22 5.3 ± 0.1 33 ± 5 30 ± 5 24 5.2 ± 0.1 35 ± 5 25 ± 5

Using the seed culture thus obtained, the transformation fermentation cycle was carried out in a vertical cylindrical stainless steel fermenter having a diameter of 1.02 m, a height of 1.5 m and a geometric volume of 1.4 m ³ . This fermenter was equipped with a turbine mixer having two impellers, one of which was located at a depth of 867 mm from the top of the reactor, and the other was located at a depth of 1435 mm from the top of the reactor. Each impeller had six blades, each of which had a radial size of 95 cm and a height of 75 cm. The vertical partitions had a height of 1440 mm and were located radially inside the reactor at a distance of 100 cm from the inner vertical wall of the fermenter. A nutrient mixture (100 L) was obtained having the composition shown in Table 11:

Table 11. Culture for biological transformation in a 1000 liter fermenter quantity g / l Glucose 16 kg 23 Peptone 16 kg 23 autolysed yeast 16 kg 23 defoamer SAG 471 0.080 kg trace amount deionized water ext. up to 700 l - sterilization of an empty fermenter for 1 hour at 130 ° C - loading of 600 l of deionized water; - heating at 40 ° C - adding, with stirring, the components of the environment - stirring for 15 minutes, - bringing to a volume of 650 l - sterilization at 121 ° C, 30 minutes - pH after sterilization = 5.7 - adding sterile deionized water up to 700 l

An initial loading (700 L) of this nutrient mixture (pH = 5.7) was introduced into the fermenter, and then the seed inoculum of this example (7 L) obtained as described above was introduced.

The inoculum-containing nutrient mixture was incubated for 24 hours at an aeration rate of 0.5 l / l / min and at a head pressure of 0.5 kg / cm ² . The temperature was maintained at 28 ° C, and the stirring speed was 110 rpm. The increase in mycelium volume is shown in Table 12, which also shows the pH and dissolved oxygen profiles in the seed culture reaction.

Table 12. Mycelium growth time in a fermenter with a fermentation culture Fermentation period, hours pH The volume of precipitated mycelium (PMV),% (3000 rpm / 5 min.) Dissolved oxygen,% 0 5.6 ± 0.2 100 4 5.5 ± 0.2 100 8 5.5 ± 0.2 12 ± 3 95 ± 5 12 15 ± 3 90 ± 5 16 5.4 ± 0.1 20 ± 5 75 ± 5 twenty 5.3 ± 0.1 25 ± 5 60 ± 5 22 5.2 ± 0.1 30 ± 5 40 ± 5

At the end of the incubation, precipitation of mycelium was observed, but this precipitate was mainly small and relatively loose. Diffuse mycelium was suspended in the broth. The final pH was 5.1-5.3.

To the culture thus obtained, a suspension of canrenone (1.250 kg; finely divided to a particle size of 5 microns) in sterile water (5 L) was added for transformation. A sterile additive solution and an antibiotic solution were added in the ratios indicated in Table 14 for reaction time 0. The composition of the additive solution is shown in Table 13.

Table 13. Additive solution (for transforming culture) quantity Dextrose 40 kg Autolysed yeast 8 kg Defoamer SAG 471 0.010 kg deionized water ext. up to 100 l - sterilization of an empty 150 l fermenter for 1 hour at 130 ° C - loading of 70 l of deionized water; heating at 40 ° C - adding, with stirring, the components of the “additive solution” - mixing for 30 minutes, bringing to a volume of 95 l - pH 4.9 - sterilization at 120 ° C × 20 minutes - pH after sterilization of about 5

Biological transformation was carried out for about 96 hours at an aeration rate of 0.5 l / l / min and a pressure in the head of 0.5 kg / cm ² , and the pH was 4.7-5.3, which was adjusted if this necessary by adding 7.5 M NaOH or ₄ M H ₃ PO ₄ . The initial aeration speed was 100 rpm, after which after 40 hours it was increased to 165 rpm, and after 64 hours to 250 rpm. The initial temperature was 28 ° C, and then, when the PMV reached 45%, the temperature was lowered to 26 ° C, and when the PMV increased to 60%, the temperature was lowered to 24 ° C. To control foaming, if necessary, a few drops of SAG 471 were added. Glucose levels during the fermentation reaction were monitored at 4-hour intervals, and when the glucose concentration dropped below 1 g / l, an additional amount of sterile additive solution was added to this batch (10 l). The disappearance of canrenone and the appearance of 11α-hydroxycanrenone were monitored during the reaction by HPLC. When at least 90% of the starting canrenone was converted to 11α-hydroxycanrenone, another 1,250 kg of canrenone was added. When 90% conversion of canrenone was detected in this added amount, another 1,250 kg of canrenone was added. Using the same criterion, additional quantities of canrenone (1.250 kg portions) were added until the entire charge (20 kg) was introduced into the reactor. After adding the total amount of canrenone, the reaction was completed when the concentration of unreacted canrenone substrate with respect to the amount of 11α-hydroxycanrenone produced was 5%. The addition of canrenone, a sterile supplement solution, and an antibiotic solution are shown in Table 14.

Table 14. Addition of steroid and solutions (additives and antibiotics) during the biological conversion of canrenone in a fermenter Reaction time, hours Canrenone in suspension Sterile additive solution, liters The solution of antibiotics, liters volume, ~ liters kg adding kg 0 1,250 1.25 10 8 700 4 10 8 1,250 2,5 10 12 10 16 1,250 10 twenty 10 24 1,250 5 10 8 800 28 1,250 10 32 1,250 10 36 1,250 10 40 1,250 10 44 1,250 10 48 1,250 12.5 10 8 900 52 1,250 10 56 1,250 10 60 1,250 10 64 1,250 10 68 1,250 10 72 1,250 twenty 10 8 1050 76 0 80 84 88 92 Total

When the biological conversion was completed, the mycelium was isolated from the broth by centrifugation in a basket centrifuge. As determined by HPLC, the filtrate contained only 2% of the total amount of 11α-hydroxycanrenone in the collection broth and was therefore removed. Then, the mycelium was suspended in ethyl acetate (1000 L) in a 2 m ³ extraction tank. This suspension was heated for one hour with stirring and under reflux with ethyl acetate, and then cooled and centrifuged in a basket centrifuge. The mycelial precipitate was washed with ethyl acetate (200 L) and then drained. The steroid-rich solvent extract was left for one hour to separate the aqueous phase. The aqueous phase was extracted with additional ethyl acetate (200 L), and then discarded. The combined solvent phases were clarified by centrifugation and placed in a concentrator (geometric volume of 500 L), and then concentrated in vacuo to a residual volume of 100 L. Upon evaporation, the initial loading of the combined extract and washing solutions into the concentrator was 100 L, and this volume was kept constant by continuously or periodically adding the combined solution as the solvent evaporated. After the evaporation stage was completed, the bottoms were cooled to a temperature of 20 ° C and stirred for two hours, and then filtered on a Buchner filter. The hub vessel was washed with ethyl acetate (20 L) and this wash solution was then used to wash the filter cake. The resulting product was dried in vacuo for 16 hours at 50 ° C. The yield of 11α-hydroxycanrenone was 14 kg.

Example 4

The lyophilized spores of Aspergillus ochraceus NRRL 405 were suspended in a culture medium with liquid corn extract (2 ml) having the composition shown in Table 15:

Table 15. Medium with liquid corn extract (Medium for cultivation of the primary seed crop) Liquid Corn Extract 30 g Yeast extract 15 g Monobasic Ammonium Phosphate 3 g Glucose (loading after sterilization),
distilled water - a sufficient amount to 1000 ml of pH 4.6, adjusted to pH 6.5 by adding 20% NaOH;
distribution of 50 ml in 250 ml Erlenmeyer flasks;
sterilization at 121 ° C for 20 minutes. 30 g

The resulting suspension was used in the inoculum for propagating spores on agar plates. Ten agar plates were prepared, each containing a solid culture medium: glucose / yeast extract / phosphate / agar having the composition shown in Table 16:

Table 16. GYPA (glucose / yeast extract / phosphate / cup agar Glucose (loading after sterilization) 10 g Yeast extract 2.5 g K ₂ HPO ₄ 3 g Agar
distilled water, a sufficient amount of up to 1000 ml, adjusting the pH to 6.5, sterilization at 121 ° C, 30 minutes 20 g

A 0.2 ml aliquot of the suspension was transferred onto the surface of each plate. These plates were incubated for ten days at 25 ° C, after which the spores from all plates were collected in a sterile cryogenic protective medium having the composition shown in Table 17:

Table 17. GYP / glycerin (glucose / yeast extract / phosphate / agar for vessels with a uterine environment) Glucose (loading after sterilization) 10 g Yeast extract 2.5 g K ₂ HPO ₄ 3 g Glycerol
distilled water, a sufficient amount of up to 1000 ml, sterilization at 121 ° C, 30 minutes 20 g

The resulting suspension was distributed into 20 vessels, with 1 ml of suspension being transferred to each vessel. These vessels contained a basic stock of cells that could be used to produce a working stock of cells in order to generate an inoculum for the biological conversion of canrenone to 11α-hydroxycanrenone. These vessels containing the main bank of cells were stored in a freezer in the vapor phase of liquid nitrogen at -130 ° C.

To start producing a working stock of cells, spores from one vessel with the original cell stock were resuspended in a sterile culture medium (1 ml) having the composition shown in Table 15. This suspension was divided into ten 0.2 ml aliquots and each aliquot was used to inoculate agar plates with a solid culture medium having the composition shown in Table 16. These plates were incubated for ten days at 25 ° C. On the third day of incubation, the lower part of the culture medium had a brown-orange color. At the end of the incubation, intensive production of spores having a golden color was observed. Spores from each dish were collected by the method described above to obtain the original cell stock. In total, one hundred vessels were prepared, each of which contained 1 ml of suspension. These vessels contained a working cell stock. Vessels with a working cell stock were also stored in a freezer in the vapor phase of liquid nitrogen at -130 ° C.

Cultivation medium (50 ml) having the composition shown in Table 15 was loaded into a 250 ml Erlenmeyer flask. An aliquot (0.5 ml) of the working cell suspension was introduced into the flask and mixed with the culture medium. The inoculated mixture was incubated for 24 hours at 25 ° C. to produce a primary seed culture having a precipitated mycelium volume of approximately 45%. After visual observation of the culture, it was found that it contains granule-like mycelium with a diameter of 1 to 2 mm; and after observation under a microscope, it was identified as a pure culture.

The cultivation of the secondary seed culture was initiated by introducing the culture medium having the composition shown in Table 15 into a 2.8 liter Fernbach flask followed by inoculation of the medium with a portion (10 ml) of the primary seed culture of this example, the preparation of which was described above. This inoculated mixture was incubated at 25 ° C. for 24 hours on a rotary shaker (200 rpm, offset 5 cm). At the end of the incubation, the culture had the same properties as described above for the primary seed culture, and this culture can be used for the transformation reaction by fermentation, in which canrenone is biologically converted to 11α-hydroxycanrenone.

The transformation was carried out in a Biostat Braun E fermenter, the description of which is presented below:

Capacity 15 liters with a round bottom Height 53 cm Diameter 20 cm Height / diameter 2.65 Impeller diameter 7.46 cm, 6 blades 2.2 × 1.4 cm each Paddle wheel arrangement 65.5, 14.5 and 25.5 cm from the bottom of the tank Partitions four; 1.9 × 48 cm Bubbler diameter 10.1 cm, 21 holes Temperature control provided by outer jacket

Canrenone at a concentration of 20 g / L was suspended in deionized water (4 L) and a portion (2 L) of the culture medium having the composition shown in Table 18 was added while stirring the mixture in a fermenter at 300 rpm.

Table 18. Growth medium for biological transformation of culture in a 10 l fermenter quantity quantity / l glucose (loading after sterilization) 160 g 20 g Peptone 160 g 20 g yeast extract 160 g 20 g defoamer SAG 471 4.0 ml 0.5 ml canrenon
deionized water ext. up to 7.5 l
- sterilization of the empty fermenter for 30 min at 121 ° C 160 g 20 g

The resulting suspension was stirred for 15 minutes, after which the volume was adjusted to 7.5 L by adding deionized water. At this stage, the pH of the suspension was adjusted to 5.2-6.5 by adding 20% of the mass. NaOH solution, after which the suspension was sterilized by heating at 121 ° C for 30 minutes in a Braun E fermenter. The pH after sterilization was 6.3 ± 0.2, and the final volume was 7.0 L. The sterilized suspension was inoculated with a portion (0.5 L) of the secondary seed culture of this example, which was obtained as described above, and the volume was adjusted to 8.0 L by adding a 50% sterile glucose solution. Fermentation was carried out at a temperature of 28 ° C until the PMV reached 50%, after which the temperature was lowered to 26 ° C, and then, when the PMV exceeded 50%, the temperature was lowered to 24 ° C to maintain a constant PMV level of approximately below 60 % Air was introduced through a bubbler at a speed of 0.5 rpm / min, based on the initial volume of the liquid, and the pressure in the fermenter was maintained at 700 mbar. Stirring was started at a speed of 600 rpm, and then, if necessary, gradually increased to 1000 rpm to maintain the level of dissolved oxygen above 30% vol. Glucose concentration was continuously monitored. After initially high glucose concentration fell below 1%, due to its absorption during the fermentation reaction, an additional amount of glucose was added in the form of a 50% sterile glucose solution to maintain its concentration within 0.05% -1% for the entire remaining cycle for this batch. Before inoculation, the pH was 6.3 ± 0.2. After lowering the pH to about 5.3 during the initial fermentation period, the pH was maintained within 5.5 ± 0.2 for the remainder of the cycle by the addition of ammonium hydroxide. Foaming was controlled by the addition of a polyethylene glycol antifoam commercially available under the trademark SAG 471 OSI Specialties, Inc.

Culture growth was observed in the first 24 hours of the cycle, during which the PMV was about 40%, the pH was about 5.6, and the dissolved oxygen content was about 50% vol. The transformation of canrenone began even during the growth of culture. Concentrations of canrenone and 11α-hydroxycanrenone were monitored during biological conversion by daily analysis of samples. Samples were extracted with hot ethyl acetate and the resulting sample solution was analyzed by TLC and HPLC. Biological conversion was considered complete if the concentration of residual canrenone was about 10% of the initial concentration. The approximate conversion time was 110-130 hours.

After completion of the biological conversion, the mycelial biomass was isolated from the broth by centrifugation. The supernatant was extracted with an equal volume of ethyl acetate, and the aqueous layer was discarded. The mycelial fraction was resuspended in ethyl acetate using approximately 65 volumes per gram of canrenone loaded into the fermentation reactor. Then the mycelial suspension was heated under reflux for one hour, stirring at the same time, and then cooled to about 20 ° C and filtered on a Buchner funnel. The mycelial filter cake was washed twice with 5 volumes of ethyl acetate per gram of canrenone loaded into the fermenter, and then washed with deionized water (1 L) to remove residual ethyl acetate. The aqueous extract, enriched solvent, solvent washing and water washing were combined. The remaining depleted mycelial precipitate was either discarded or extracted again, depending on the result of the analysis for the presence of residual steroids in it. The combined liquid phases were left for two hours to settle. And the organic phase was concentrated in vacuo until the residual volume became approximately 500 ml. Then the vessel of the apparatus was cooled to about 15 ° C. by slow stirring for about one hour. The crystalline product was isolated by filtration and washed with chilled ethyl acetate (100 ml). The solvent was removed from the crystals by evaporation and the crystalline product was dried in vacuo at 50 ° C.

Example 5

Lyophilized spores of Aspergillus ochraceus ATCC 18500 were suspended in a culture medium with liquid corn extract (2 ml) as described in Example 4. Ten agar plates were obtained by the method also described in Example 4. These plates were incubated and spores were collected as described in Example 4 for receiving the basic cell fund. Vessels containing the main cell stock were stored in the freezer in the vapor phase of liquid nitrogen at -130 ° C.

A working cell fund was obtained from a vessel with a basic cellular fund, as described in Example 4, and stored in a freezer in liquid nitrogen at -130 ° C.

Cultivation medium (300 ml) having the composition shown in Table 19 was loaded into a 2 L septum flask. An aliquot (3 ml) of the working cell suspension was introduced into this flask. The inoculated mixture was incubated for 20-24 hours at 28 ° C on a rotating shaker (200 rpm, offset 5 cm) with the production of the primary seed culture, which had a sedimented mycelium volume of approximately 45%. After a visual inspection, it was found that this culture contains granule-like mycelium with a diameter of granules of 1-2 mm; and after observation under a microscope, it was found that this culture is pure.

Table 19. Primary and Secondary Seed Culture Glucose (loading after sterilization) 20 g Peptone 20 g Yeast extract 20 g distilled water, a sufficient amount of up to 1000 ml, sterilization at 121 ° C, 30 minutes

Cultivation of the secondary seed culture was initiated by introducing 8 L of culture medium having the composition shown in Table 19 into a 14 liter glass fermenter. This fermenter was inoculated with 160-200 milliliters of the primary seed culture of this example. The receipt of this culture is described above.

The inoculated mixture was cultured at 28 ° C for 18-20 hours with stirring at a speed of 200 rpm and at an aeration speed of 0.5 rpm / min. At the end of cultivation, the culture had the same properties as the primary culture described above.

The transformation was carried out in a 60 L fermenter, mainly by the method described in Example 4, except that the culture medium had the composition shown in table 20, and the initial load of the secondary seed culture was 350-700 ml. The stirring speed was initially 200 rpm, but then it was increased to 500 rpm because of the need to maintain the level of dissolved oxygen above 10% vol. The approximate biological conversion time for 20 g / L canrenone was 80-160 hours.

Table 20. Growth medium for biological transformation of culture in a 60 l fermenter quantity quantity / l glucose (loading after sterilization) 17.5 g 0.5 g Peptone 17.5 g 0.5 g yeast extract 17.5 g 0.5 g Canrenone (loading as a 20% suspension in sterile water) 700 ml 20 g sufficient amount of deionized water up to 35 l, sterilization for 30 min at 121 ° C

Example 6

Using a spore suspension from the working cell stock produced by the method described in Example 4, primary and secondary seed cultures were obtained, also mainly by the method described in Example 4. Two cycles were carried out using the secondary seed culture produced by this method biological transformation in accordance with the modified method illustrated in FIG. 1, and two cycles of biological transformation were carried out in the manner illustrated in FIG. 2. The culture medium for transformation, the canrenone addition scheme, the collection time and the degree of conversion for these cycles are shown in Table 21. In the R2A cycle, the canrenone addition scheme, mainly described in Example 3, was used, while the modified scheme of Example 3 was used in the R2C cycle, only with with two canrenone supplements, one at the beginning of the cycle and the other after 24 hours. In cycles R2B and R2D, the entire canrenone charge was introduced at the beginning of the cycle, and this process was carried out mainly by the method described in Example 4, except that before the canneron was introduced into the fermenter, the canrenone charge was sterilized in a separate vessel and glucose was added as the reaction progressed this party. To minimize the formation of lumps formed after sterilization, a Waring homogenizer was used. In cycles R2A and R2B, canrenone was introduced into the batch in methanol solution and, in this regard, these cycles also differed from the cycles described in Examples 3 and 4, respectively.

In cycles R2A and R2B, the concentration of methanol in the fermented medium accumulated up to about 6.0% and this concentration was found to inhibit culture growth and biological conversion. However, based on the results of these cycles, it was concluded that methanol or another water-miscible solvent can be successfully used at lower concentrations to increase the loading of canrenone and to ensure the presence of canrenone in the form of a finely divided precipitate, which leads to an increase in the interface area and promotes the delivery of canrenone to the reaction zone.

Canrenone is stable at sterilization temperature (121 ° C), but forms lumps. For crushing these lumps into small particles, a Waring homogenizer is used, which contributes to the successful conversion of canrenone into the desired product.

Example 7

Using a spore suspension from the working cell stock produced by the method described in Example 4, primary and secondary seed cultures were obtained, also mainly by the method described in Example 4. The description and results of Example 7 are presented in Table 22. Using a secondary seeded culture produced in this way, one cycle of biological transformation (R3C) was carried out mainly by the method described in Example 3, and three cycles of biological transformation was carried out by the method described in general Example 5. In the last three cycles (R3A, R3B and R3D), canrenone was sterilized in a portable reservoir along with a glucose-free culture medium. Glucose was supplied from another reservoir under aseptic conditions. The sterilized suspension of canrenone was introduced into the fermenter either before inoculation or during the early stage of biological conversion. In the R3B cycle, additional sterile canrenone and culture medium were added after 46.5 hours. Lumps of canrenone formed after sterilization were crushed using a Waring homogenizer, which contributed to the production of a finely dispersed suspension introduced into the fermenter. The transforming medium for cultivation, the scheme for adding canrenone, the scheme for adding nutrients, the collection time and the degree of conversion for these cycles are presented in Tables 22 and 23.

Due to the growth of filaments, a highly viscous broth was observed in the fermenter in all four cycles of this Example. In order to remove the obstacles created by the high viscosity with respect to aeration, mixing, pH adjustment and temperature control, the aeration rate and the stirring speed were increased during these cycles. The transformation reactions took place satisfactorily even under more severe conditions, but a dense precipitate formed on the surface of the liquid broth. Due to this precipitate, a certain amount of unreacted canrenone was removed from the broth.

Example 8

The description and results of Example 8 are systematized in Table 24. Four fermentation cycles were carried out in which 11α-hydroxycanrenone was produced by biological conversion of canrenone. In two of these cycles (R4A and R4D), biological conversion was carried out basically in the same way as was described for the R3A and R3D cycles in Example 6. In the R4C cycle, canrenone was converted to 11α-hydroxycanrenone, basically the same by the method described in Example 3. In the R4B cycle, the process was carried out mainly by the method described in Example 4, that is, with the sterilization of canrenone and culture medium in a fermenter immediately before inoculation; all nitrogen and phosphorus nutrients were introduced at the beginning of the cycle, and an additional solution containing only glucose was introduced into the fermenter to maintain glucose levels as the cycle progressed. In the latter process (R4B cycle), glucose concentration was monitored every 6 hours and a glucose solution was added in the indicated amount to maintain glucose levels in the range of 0.5 to 1%. Schemes for the addition of canrenone for these cycles are presented in Table 25.

In all fermenters, high-speed stirring and aeration were carried out for most of the fermentation cycle, since the fermented medium became highly viscous one day later after inoculation.

Example 9

Culturing transforming media, canrenone addition schemes, collection times and conversion rates for cycles in this Example are shown in Table 26.

Four biological conversion cycles were carried out mainly by the method described for the R4B cycle in Example 8, with the exception of the procedures described below. In cycle R5B, the upper impeller of a turbine disk mixer used for mixing in other cycles was replaced by a marine rotor for pumping from top to bottom. The action of pumping from top to bottom provides axial flow of the broth to the center of the fermenter and helps to reduce the formation of dense sediment. Methanol (200 ml) was added immediately after inoculation in the R5D cycle. Since canrenone was sterilized in the fermenter, all nutrients, with the exception of glucose, were fed at the beginning of the cycle, which avoided the need for a chain supply of nitrogen sources, phosphorus sources, or antibiotics.

To ensure the wetting of the solid phase protruding above the surface of the liquid, culture medium (2 L) was added to each fermenter 96 hours after the start of the cycle. The problems associated with stirring can, in general, be overcome either by adding a culture medium or by using the pump impeller with pumping from top to bottom (cycle R5B), but the results of these cycles indicate the feasibility and advantages of this method and indicate that sufficient stirring can be carried out in accordance with standard practice.

Example 10

Three cycles of biological conversion were carried out mainly by the method described in Example 9. Transforming media for cultivation, canrenone addition schemes, collection times and conversion rates for the cycles in this Example are presented in Table 27.

Cultivation medium (1.3 L) and sterile water (0.8 L) were added after 71 hours in the R6A cycle to impregnate a dense precipitate of mycelium protruding above the surface of the liquid broth. For the same purposes, culture medium (0.5 L) and sterile water (0.5 L) were added after 95 hours in the R6B cycle. Data on the equilibrium of materials showed that mass equilibrium is better determined when the level of dense sediment protruding above the surface of the liquid is minimized.

Example 11

Fermentation cycles were conducted to compare pre-sterilization of canrenone with sterilization of canrenone and culture medium in a transformation fermenter. The R7A cycle was carried out by the method illustrated in FIG. 2 under conditions comparable to the conditions of the cycles R2C, R2D, R3A, R3B, R3D, R4A and R4D. Cycle R7B is illustrated in FIG. 3 under conditions comparable to the conditions of the cycles conducted as described in Examples 4, 9 and 10 and of the cycle R4B. Culturing transforming media, canrenone addition schemes, collection times, and conversion rates for cycles in this Example are shown in Table 28.

Table 28. Description of the method for conducting an experiment on 10 l of large-scale biological transformation Cycle number R7A R7B Wednesday (g / l) cycle similar to cycle R7A liquid corn extract thirty yeast extract fifteen NH ₄ H ₂ PO ₄ 3 glucose fifteen OSA 0.5 ml pH adjusted to 6.5 by adding 2.5 N NaOH Loading canrenone 160 g of canrenone was sterilized and stirred outside the fermenter 160 g of canrenone was sterilized in a fermenter Loading environment Glucose supply; canrenone was added with 1.6 L of culture medium Glucose supply; no other substances were added Pick time 118, 5 hours 118.5 hour Biological transformation 93% 89%

The mass balance determined from the final sample taken from the R7B batch was 83%, which indicated that no significant loss or decomposition of the substrate was observed during biological conversion. It was found that stirring for both cycles was adequate.

The residual glucose concentration exceeded the control range, preferably by 5-10 g / liter in the first 80 hours of the reaction. The loose sediment that accumulated in the head of both fermenters did not have any noticeable adverse effect on the cycle productivity.

Example 12

The extraction efficiency was determined in a series of 1 L extraction cycles and systematized in Table 29. In each of these cycles, steroids were extracted from mycelium using ethyl acetate (fermentation volume 1 L / L). Two successive extractions were performed in each cycle. Based on RP-HPLC, about 80% of the total steroid was isolated during the first extraction; and during the second extraction, the steroid yield increased to 95%. At the third extraction, another 3% steroid was to be isolated. The remaining 2% of the steroid was the loss in the aqueous phase of the supernatant. The extract was evaporated to dryness in vacuo, but was not washed with any additional solvent. Solvent treatment should improve the initial extraction yield in terms of economic costs.

Table 29. Isolation of 11α-hydroxycanrenone upon 1 liter extraction (% total) Cycle number 1st extract 2nd extract 3rd extract Supernatant R5A 79% 16% 2% 2% R5A 84% 12% 2% 2% R4a 72% 20% 4% 4% R4a 79% 14% 2% 5% R4b 76% 19% 4% 1% R4b 79% 16% 3% 2% R4b 82% fifteen% 2% 1% Average 79% 16% 3% 2%

Methylisobutylketone (MIBK) and toluene were evaluated as extraction / crystallization solvents for 11α-hydroxycanrenone at a scale of 1 L of broth. Using the above extraction scheme, MIBK and toluene were comparable with ethyl acetate in both extraction efficiency and crystallization productivity.

Example 13

As a partial evaluation of the methods illustrated in FIGS. 2 and 3, particle size testing was performed using a canrenone substrate added at the beginning of the fermentation cycle in each of these methods. As described above, in the method illustrated in FIG. 1, canrenone was finely ground but not sterilized before being introduced into the fermenter, while the growth of undesirable microorganisms was suppressed by the addition of antibiotics. In the methods illustrated in FIGS. 2 and 3, canrenone was sterilized before the reaction. In the method illustrated in FIG. 2, canrenone was sterilized in a mixer before being introduced into the fermenter. In the method illustrated in FIG. 3, a suspension of canrenone in the culture medium was sterilized in a fermenter at the beginning of the cycle. As discussed above, sterilization can lead to agglomeration of canrenone particles. Due to the limited solubility of canrenone in an aqueous medium for cultivation, the productivity of the method depends on mass transfer from the solid phase, and therefore it can be expected to depend on the interfacial surface area determined by the surface area of the particulate solid substrate, which, in turn, depends on the particle size distribution composition of these particles. These considerations serve mainly as constraints in the methods illustrated in FIGS. 2 and 3.

However, it was found that stirring in the mixer, Figure 2, and in the fermentation tank, Figure 3, together with the action of the shear pump used to feed the batch, Figure 2, contributes to the destruction of these agglomerates into particles with a size sufficient to so that you can supply unsterilized and finely ground canrenone in the cycle shown in figure 1. This was illustrated by evaluating the particle size distribution for the canrenone particles present at the beginning of the reaction cycle of each of the three methods. See Table 30 and Figs. 4 and 5.

Table 30. Particle distribution for three different canrenone samples Sample 45-125 μm <180 μm average size, microns # lot:% biological conversion Loading canrenone 75% 95% - R3C:
93.1% (120 h)
R4C:
96.3% (120 h) Mixed sample 31.2% 77.2% 139.5 R3A:
94.6% (120 h)
R4B:
95.2% (120 h) Sterilized sample 24.7% 65.1% 157.4 R4B:
97.6% (120 h)
R5B:
93.8% (120 h)

Based on the data in Table 30, it should be noted that mixers and a shear pump are effective in reducing the average particle size of sterilized canrenone to the same order of magnitude that is characteristic of an unsterilized substrate, but at the same time, a significant difference in particle sizes remains , which testifies in favor of an unsterilized substrate. Despite this difference, the reaction productivity data indicate that the pre-sterilization method is at least as productive as the method illustrated in FIG. 1. Additional advantages can be realized in the method illustrated in FIG. 2, using several steps to further reduce and control the particle size, for example by wet grinding sterilized canrenone and / or by pasteurization instead of sterilization.

Example 14

A seed culture was obtained by the method described in Example 5. After 20 hours, the mycelium in the inoculum fermenter had a soft consistency with 40% PMV. Its pH was 5.4, and 14.8 g / L glucose was left untreated.

A culture transforming medium (35 L) was obtained having the composition shown in Table 20. To obtain a nutrient medium, glucose and yeast extract were sterilized separately and mixed to obtain a uniform nutrient mass with an initial concentration of 30% by weight. glucose and 10% of the mass. yeast extract. The pH of the nutrient mass was adjusted to 5.7.

Using this medium (Table 20), two cycles of biological conversion of canrenone to 11α-hydroxycanrenone were carried out. Each of these cycles was carried out in a 60 liter fermenter equipped with a mixer comprising one Rushton turbine impeller and two Lightnin A315 turbine impellers.

The initial loading of the culture medium into the fermenter was 35 L. Finely ground and unsterilized canrenone was added to an initial concentration of 0.5%. The medium in the fermenter was inoculated with a seed culture prepared as described in Example 5 with an initial degree of inoculation of 2.5%. Fermentation was carried out at a temperature of 28 ° C, with a stirring speed of 200-500 rpm, with an aeration speed of 0.5 rpm / min and with back pressure sufficient to maintain the level of dissolved oxygen at least 20% vol. The culture for transformation, developing during the production cycle, had the form of very small oval granules (about 1-2 mm). Canrenone and additional nutrients were introduced into the fermenter by chain feeding, mainly by the method described in Example 1. Nutrient additives were introduced every four hours in the ratio of 3.4 g of glucose and 0.6 g of yeast extract per liter of broth in the fermenter.

Table 31 shows the aeration rate, stirring rate, dissolved oxygen level, PMV and pH, determined at the appropriate time intervals for each of the cycles described in this example, as well as the scheme for adding glucose during this cycle. Table 32 shows the canrenone conversion profile. The R11A cycle completed after 46 hours; R11B cycle lasted for 96 hours. In the last cycle, 81% conversion was achieved in 81 hours; after 84 hours, another supplementation of the nutrient mixture was carried out, after which the supply of nutrients was completed. It should be noted that a significant change in viscosity occurred in the period between the termination of the supply of nutrients and the end of the cycle.

Example 15

Various cultures were tested for the effectiveness of the biological conversion of canrenone to 11α-hydroxycanrenone in a manner substantially described above.

A working cell stock for each of the microorganisms Aspergillus niger ATCC 11394, Rhizopus arrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b was obtained by the method described in Example 5. A culture medium (50 ml) having the composition shown in Table 18 was inoculated with a spore suspension (1 ml) from the working cell stock and placed in an incubator. A seed culture was obtained in an incubator by fermentation for 20 hours at 26 ° C. The contents of the incubator were mixed at a speed of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used to inoculate transformation flasks containing the culture medium (30 ml) shown in Table 18. Each culture was used to inoculate two flasks, with a total of six flasks. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36 ° C and 0.5 ml aliquots of this solution were introduced into each of these flasks. Biological conversion was carried out mainly under the conditions described in Example 5, with daily addition of 50% of the mass. glucose solution (1 ml). After the first 72 hours, mycelium growth was monitored in the corresponding fermentation flasks for transformation:

ATCC 11394 - good uniform growth;

ATCC 11145 - good growth in the first 48 hours, however, aggregation of mycelium into lumps was observed; growth was not observed in the last 24 hours;

ATCC 6227b - good growth; the mycelial mass is aggregated into lumps.

Samples of the broth were analyzed for the degree of biological conversion. Three days later, fermentation using ATCC 11394 resulted in 80-90% conversion to 11α-hydroxycanrenone; ATCC 11145 gave a 50% conversion; and ATCC 6227b gave a conversion of 80 to 90%.

Example 16

Using the method mainly described in Example 15, other microorganisms were tested for the conversion efficiency of canrenone to 11α-hydroxycanrenone. The tested microorganisms and test results are presented in Table 33.

Example 17

Various microorganisms were tested for the effectiveness of the conversion of canrenone to 9α-hydroxycanrenone. Fermentation media that were obtained for the fermentation cycles of this Example are presented in Table 34.

Table 34. Soya flour: Dextrose 20 g soy flour 5 g NaCl 5 g yeast extract 5 g KN ₂ PO ₄ 5 g Water up to 1 l pH 7.0 Peptone / Yeast Extract / Glucose: Glucose 40 g Bactopeptone 10 g Yeast extract 5 g Water up to 1 l Miller-Hinton Wednesday: Meat broth 300 g Casino acids 17.5 g Starch 1.5 g Water up to 1 l

Mushrooms were grown in a medium with soy flour and in a medium based on peptone-yeast extract-glucose; actinomycetes and eubacteria were grown in a medium with soy flour (+ 0.9% wt. Na formate for biological transformations) and in Miller-Hinton broth.

Stock cultures were inoculated with freshly frozen spore stock cultures (20 ml of soy flour in a 250 ml Erlenmeyer flask). The flasks were coated with a frosted glass filter and bioprotective coating. Initial cultures (24- or 48-hour) were used to inoculate metabolic cultures (also 20 ml in a 250 ml Erlenmeyer flask) with a 10% -15% crossing volume, then incubated for 24-48 hours and a steroid substrate was added for the transformation reaction.

Canrenone was dissolved / suspended in methanol (20 mg / ml), the filter was sterilized and added to the cultures to a final concentration of 0.1 mg / ml. All flasks for the conversion reaction by fermentation were shaken at 250 rpm (amplitude 2 ") at a controlled room temperature of 26 ° C and a humidity of 60%.

Biological transformation products were collected at 5 and 48 hours or 24 hours after addition of the substrate. Collection began by adding ethyl acetate (23 ml) or methylene chloride to a fermentation flask. The flasks were then shaken for two minutes and the contents of each flask were poured into a 50 ml conical tube. To separate the phases, these tubes were centrifuged at 4000 rpm for 20 minutes at room temperature. The organic layer from each tube was transferred to a 20 ml borosilicate glass vessel and evaporated in a high-speed vacuum evaporator. The vessels were covered and stored at -20 ° C.

To obtain material for structural determination, the scale of biological transformations was increased to 500 ml by increasing the number of shaker flasks for fermentation to 25. At the time of collection (24 or 48 hours after adding the substrate), ethyl acetate was added separately to each flask and the flasks were covered and placed back to the shaker for 20 minutes. Then the contents of the flasks were embroidered in polypropylene bottles and centrifuged to separate the phases, or poured into a separation funnel in which the phases were separated by gravity. The organic phase was dried to give a crude steroid extract contained in the reaction mixture.

The reaction product was first analyzed by thin layer chromatography on inverted fluorescence plates with silica gel (254 μm). Ethyl acetate (500 μl) was added to each vessel containing the dried ethyl acetate extract from the reaction mixture. Subsequent analysis was performed using high resolution liquid chromatography and mass spectrometry. TLC plates were shown in a solvent mixture, chloroform / methanol, 95: 5 v / v.

Another analysis was performed using high resolution liquid chromatography and mass spectrometry. For this, Waters HPLC, Millennium software, a photodiode array detector, and an autosampler were used. In reverse phase HPLC, a Waters NovaPak C-18 column (particle size 4 μm) with a 4 mm Radial-Pak cartridge was used. Chromatography was started with a linear gradient of the solvent (25 minutes) on a column initialized in water: acetonitrile (75:25), and ended in water: acetonitrile (25:75). Then, a gradient of up to 100% acetonitrile (3 minutes) and a 4-minute iso wash were used, after which the column was regenerated under initial conditions.

For LC / MS, ammonium acetate was added to both the acetonitrile and aqueous phases at a concentration of 2 nM. This had no significant effect on chromatography. The column eluate was separated 22: 1, with most of the material being sent to a PDA detector. The remaining 4.5% of the material was sent to the chamber of a Sciex API III mass spectrometer for electrospray ionization. Mass spectrometry was carried out in a positive mode. A similar series of data obtained by HPLC chromatogram at a single wavelength using a PDA detector was transferred to a mass spectrometer for the joint analysis of UV and MS data.

Samples of mass spectrometric fragmentation can be used for selection from hydroxylated substrates. Two alleged hydroxylated canrenones, 11α-hydroxy and 9α-hydroxy, lose water at different frequencies and to an appropriate degree, which can be used as a diagnostic. In addition, 9α-hydroxycanrenone formed an ammonium adduct more easily than 11α-hydroxycanrenone. Table 35 summarizes the data from TLC, HPLC / UV and LC / MS for the fermentation of canrenone and shows which of the tested microorganisms are effective for the biological conversion of canrenone to 9α-hydroxycanrenone. Of these, the preferred microorganism is Corynespora cassiicola ATCC 16718.

Example 18

Various cultures were tested for the effectiveness of the biological conversion of canrenone to 11α-hydroxycanrenone in the manner outlined above.

The working cell stock of each of the microorganisms Aspergillus ochraceus NRRL 405 (ATCC 18500), Aspergillus niger ATCC 11394, Aspergillus nidulans ATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b, Trichothecium Protein ATCC 6287b, described in the main method, 885 described above, 4. The culture medium (50 ml) having the composition shown in Table 18 was inoculated with a spore suspension (1 ml) from the working cell stock and placed in an incubator. A seed culture was obtained in an incubator by fermentation for about 20 hours at 26 ° C. The contents of the incubator were mixed at a speed of 200 rpm.

Aliquots (2 ml) of the seed culture of each microorganism were used to inoculate transformation flasks containing the culture medium (30 ml) shown in Table 15. Each culture was used to inoculate two flasks from all 16 flasks. Androstenedione (300 mg) was dissolved in methanol (6 ml) at 36 ° C and 0.5 ml aliquots of this solution were introduced into each of these flasks. Biological conversion was carried out mainly under the conditions described in Example 6 for 48 hours. After 48 hours, the broth samples were combined and extracted with ethyl acetate as described in Example 17. Ethyl acetate was concentrated by evaporation and the samples were analyzed by thin-layer chromatography to determine if this product had chromatographic mobility similar to that of standard 11α-hydroxyandrostenedione (Sigma Chemical Co., St .Louis). The results are presented in Table 36. Positive results are indicated by "+".

Table 36. Biological conversion of androstenedione to 11α-hydroxyandrostenedione Culture ATCC # Wednesday TLC results Rhizopus oryzae 11145 CSL + Rhizopus stolonifer 6227b CSL + Aspergillus nidulans 11267 CSL + Aspergillus niger 11394 CSL + Aspergillus ochraceus NRRL 405 CSL + Aspergillus ochraceus 18500 CSL + Trichothecium roseum 12519 CSL + Trichothecium roseum 8685 CSL +

The data in Table 36 indicate that each of the listed cultures is capable of producing a compound from androstenedione having the same Rf value as standard 11α-hydroxyandrostenedione.

, размером 10×20, толщиной 250 микрон. Использовали смесь растворителей хлороформ:метанол 95:5 об/об. Кристаллизованный продукт и стандартный 11α-гидроксиандростендион анализировали с помощью ЖХ-МС и ЯМР-спектроскопии. Оба соединения имели аналогичные профили и молекулярные массы.Aspergillus ochraceus NRRL 405 (ATCC 18500) was again tested by the method described above, and culture products were isolated and purified by column chromatography on normal silica gel phase using methanol as solvent. Fractions were analyzed by thin layer chromatography. TLC plates were Whatman plates with silica gel K6F 60

, 10 × 20 in size, 250 microns thick. A solvent mixture of chloroform: methanol 95: 5 v / v was used. The crystallized product and standard 11α-hydroxyandrostenedione were analyzed by LC-MS and NMR spectroscopy. Both compounds had similar profiles and molecular weights.

Example 19A

Various microorganisms were tested for the effectiveness of the biological conversion of androstenedione to 11α-hydroxyandrostenedione in the manner generally described above in Examples 17 and 18.

Cultures of each of the microorganisms Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC 16888 and ATCC 26693, Epicoccum oryzae ATCC 7156, Curvularia lunata ATCC 12017, Cunninghamella blakesleeana ATCC 8688a and Pithomyces IFO were cultivated in 17 ways. fermentations (30 ml) had the composition shown in Table 34.

11α-Hydroxylation of androstenedione with the above microorganisms was analyzed using mainly methods similar to the product identification methods described in Examples 17 and 18. The results are presented in table 19A-1.

Table 19A-1. 11β-hydroxylation of androstenedione with various microorganisms Organism TCX LC / MS Aspergillus fumigatus ATCC 26934 + + Aspergillus niger ATCC 16888 and ATCC 26693 + + Epicoccum oryzae ATCC 7156 + + Curvularia lunata ATCC 12017 + + Cunninghamella blakesleeana ATCC 8688a + + Pithomyces atro-olivaceus IFO 6651 + +

In Table 19A-1, “+” indicates a positive result, that is, Rf has the expected value in thin-layer chromatography and approximately the corresponding molecular weight in LC / MS.

These results indicate that these microorganisms are capable of 11β-hydroxylation of androstenedione.

Example 19B

Various microorganisms were tested for the effectiveness of the biological conversion of mexrenone to 11β-hydroxymexrenone. Fermentation media for this example were prepared as described in Table 34.

The fermentation conditions and analytical methods were similar to those described in Example 17. TLC plates and a solvent system are described in Example 18. The principle of chromatographic analysis was as follows: 11α-hydroxymexrenone and 11α-hydroxycanrenone had the same chromatographic mobility, 11α-hydroxycanrenone and 9α- hydroxycanrenone had the same mobility as 11α-hydroxyandrostenedione and 11β-hydroxyandrostenedione. Therefore, 11β-hydroxymexrenone must have mobility similar to that of 9α-hydroxycanrenone. Therefore, compounds extracted from the culture medium were tested in comparison with standard 9α-hydroxycanrenone. The results are presented in Table 37.

Table 37. Systematized TLC data for the formation of 11β-hydroxycanrenone from mexrenone Microorganism Wednesday ¹ Spot character ² Absidia coerula ATCC 6647 M, S bright Aspergillus niger ATCC 16888 S, P weak (S)? (R) Beauveria bassiana ATCC 7159 R bright Beauveria bassiana ATCC 13144 S, p ?,? Botryosphaeria obtusa IMI 038560 weak Cunninghamella blakesleeana ATCC 8688a S, p bright echinulata ATCC 3655 S, p bright elegans ATCC 9245 S, p bright Curvularia lunata ATCC 12017 S bright Gongronella butleri ATCC 22822 S, p bright Penicillium patulum ATCC 24550 S, p bright Penicillium purpurogenum ATCC 46581 S, p bright Pithomyces atro-olivaceus IFO 6651 S, p weak Rhodococcus equi ATCC 14887 M weak Saccharopolyspora erythaea ATCC 11635 M, SF weak Streptomyces hygroscopicus ATCC 27438 M, SF bright Streptomyces purpurascens ATCC 25489 M, SF weak Thamnidium elegans ATCC 18191 S, p weak Thamnostylum piriforme ATCC 8992 S, p weak Trichothecium roseum ATCC 12543 P, S weak (P)? (S) ¹ M = Miller-Hinton Medium
P = PYG (peptone / yeast extract / glucose)
S = Soya Meal
SF = soy flour + formate
² ? = controversial difference from control without substrate

These data suggest that most of the microorganisms listed in this table produce a product similar or identical to 11α-hydroxymexrenone formed from mexrenone.

Example 19C.

Various microorganisms have been tested for their efficacy in converting mexrenone to 11α-hydroxymexrenone, Δ ^1,2- mexrenone, 6β-hydroxymexrenone, 12β-hydroxymexrenone and 9α-hydroxymexrenone. Mexrenone can be prepared by the method described by Weier in US Pat. No. 3,787,396 and RMWeier et al., J. Med. Chem., Vol. 18, pp. 817-821 (1975), which are incorporated herein by reference. Fermentation media were prepared as described in Example 17, except that mexrenone was included. The fermentation conditions were basically similar to the conditions described in Example 17; analytical methods were also similar to the methods described in Examples 17 and 18. TLC plates and a solvent system were similar to those described in Examples 17 and 18.

The tested microorganisms and the results obtained on their basis are presented in Table 19C-1.

Table 19C-1. The production of 11α-hydroxymexrenone from mexrenone by various microorganisms Microorganism TCX HPLC m / z 417: 399 Beauveria bassiana ATCC 7159 + + 5: 1 Beauveria bassiana ATCC 13144 + + 10: 1 Mortierella isabella ATCC 42613 + + 1: 1 Cunninghamella blakesleeana ATCC 8688a + + 1: 1 Cunninghamella echinulata ATCC 3655 + + 1: 2 Cunninghamella elegans ATCC 9245 + + 1: 1 Absidia coerula ATCC 6647 + + 1: 1 Aspergillus niger ATCC 16888 + + 4: 1 Gongronella butieri ATCC 22822 + + 3: 1 Pithomyces atro-olivaceus ATCC 6651 + + 3: 1 Streptomyces hydroscopicus ATCC 27438 + + 3: 1

In Table 19C-1, “+” indicates a positive result, that is, R _f has the expected value by thin layer chromatography and the approximate retention time by HPLC, m / z 417: 399 indicates the ratio of the peak heights of molecule 417 (hydroxymexrenone) and molecule 399 (mexrenone). The standard has a peak height ratio of 10: 1 for m / z 417 - m / z 399.

The product obtained from Beauveria bassiana ATCC 13144 was isolated from the incubation mixture and analyzed by NMR, which showed that the structural profile of this product corresponds to 11α-hydroxymexrenone. Similarly, products obtained from other microorganisms listed in Table 19C-1 are also presumably 11α-hydroxymexrenone.

Table 19C-2. The production of Δ ^1,2- mexrenone from mexrenone by various microorganisms Microorganism m / z 399 HPLC TCX Rhodococcus equi ATCC 148875 + + + Bacterium cyclo-oxydans ATCC 12673 + + + Comomonas testosteroni ATCC 11996 + + + Nocardia aurentia ATCC 12674 + + + Rhodococcus equi ATCC 21329 + + +

In Table 19C-2, “+” indicates a positive result, that is, R _f has the expected value with thin-layer chromatography and approximately the expected retention time with HPLC and the like.

The product obtained from Bacterium cyclooxydans ATCC 12673 was isolated from the incubation mixture and analyzed by NMR, which showed that the structural profile of this product corresponds to Δ ^1,2- mexrenone. Similarly, products obtained from other microorganisms listed in Table 19C-2 are also presumably Δ ^1,2- mexrenone.

The production of 6β and 12β-hydroxymexrenone

Mortierella isabella ATCC 42613 was grown as described in Example 17 in the presence of mexrenone. Fermentation products were isolated and purified using flash chromatography. Purified products were analyzed using LC / MS, as described in Examples 17 and 18, and using proton NMR and ¹³ C-NMR. Data from these analyzes indicated that these products included 6β- and 12β-hydroxymexrenone.

Table 19C-3. The production of 9α-hydroxymexrenone from mexrenone by various microorganisms Microorganism m / z 417 HPLC TCX Streptomyces hydroscopicus ATCC 27438 + + + Gongronella butieri ATCC 22822 + + + Cunninghamella blakesleeana ATCC 8688a + + + Cunninghamella echinulata ATCC 3655 + + + Cunninghamella elegans ATCC 9245 + + + Mortierella isabellina ATCC 42613 + + + Absidia coerula ATCC 6647 + + + Beauveria bassiana ATCC 7159 + + + Beauveria bassiana ATCC 13144 + + + Aspergillus niger ATCC 16888 + + +

The microorganisms listed in Table 19C-3 were grown under the same conditions as described in Example 17 in the presence of mexrenone. Fermentation products were analyzed by TLC and LC / MS as described in Examples 17 and 18. In Table 19C-3, “+” indicates a positive result, that is, R _f has the expected value in thin-layer chromatography and approximately the expected retention time by HPLC and etc. These data suggest that the products obtained include 9α-hydroxymexrenone.

Example 19D

Various microorganisms have been tested for their effectiveness in converting canrenone to Δ ^9.11 -canrenone. Fermentation media and culturing conditions were basically the same as in Example 17, except that canrenone was included in this medium. The analysis methods were similar to the methods described in Example 17 and 18. Microorganisms and the results are presented below in Table 19D-4.

Table 19D-4. The production of Δ ^9,11 -canrenone from canrenone by various microorganisms Microorganism m / z 339 HPLC TCX Bacterium cyclo-oxydans ATCC 12673 + + + Comomonas testosteroni ATCC 11996 + + + Cylindrocarpon radicicola ATCC 11011 + + + Paecilomyces carneus ATCC 46579 + + + Septomyxa affinis ATCC 6737 + + + Rhodococcus spp. ATCC 19070 + + +

Fermentation products were analyzed using TLC and LC / MS as described in Examples 17 and 18. The symbol “+” indicates a positive result, that is, R _f has the expected value for thin-layer chromatography and approximately the expected retention time by HPLC, etc. .

The product obtained from Comomonas testosteroni ATCC 11996 was isolated from the culture medium and analyzed by UV spectroscopy. The spectroscopic profile confirmed the presence of Δ ^9,11 -canrenone. Similarly, products obtained from the microorganisms listed above in Table 19D-1 were also presumably Δ ^9.11 -canrenone.

Example 20A

Scheme 1: Step 1: Method A: Preparation of 5'R (5'α), 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'a, 13'α-dimethyl-3 ', 5-di -oxospiro [furan-2 (3H), 17'α (5'H) - [7,4] metheno [4H [cyclopenta- [a] phenanthrene] -5'-carbonitrile

To a 50 gallon enameled reactor (189.25 dm ³ ), 61.2 L (57.8 kg) of DMF was loaded with stirring, followed by 23.5 kg of 11-hydroxycanrenone 1. 7.1 kg of lithium chloride was added to this mixture. The mixture was stirred for 20 minutes and 16.9 kg of cyanohydrin acetone was charged, followed by 5.1 kg of triethylamine. The resulting mixture was heated to 85 ° C and maintained at this temperature for 13-18 hours. After completion of the reaction, 353 L of water was added, followed by 5.6 kg of sodium bicarbonate. The mixture was cooled to 0 ° C, transferred to an enameled reactor with a capacity of 200 gallons (757 dm ³ ) and slowly quenched using 130 kg of a 6.7% sodium hypochlorite solution. The product was filtered and washed with 3 × 40 L portions of water to obtain 21.4 kg of enamine product.

H ¹ NMR (DMSO-d ₆ ): 7.6 (2H, brd), 4.53 (1H, d, J = 5.9), 3.71 (1H, m), 3.0-1 3 (17H, m), 1.20 (5H, m), 0.86 (3H, s), 0.51 (1H, t, J = 10).

Example 20B Preparation of 7α-cyano-11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-21-carboxylic acid, γ-lactone

50.0 g of 11-hydroxycanrenone and 150.0 ml of dimethylacetamide were added to a clean, dry, three-necked flask equipped with a mechanical stirrer, a refrigerator, a thermocouple and a housing for heating. To this mixture was added 16.0 ml of a sulfuric acid solution (obtained by mixing 50.0 ml of sulfuric acid (98.7% Baker acid) with 50.0 ml of water). Then a sodium cyanide solution containing 15.6 g of sodium cyanide and 27.0 ml of water was added.

The resulting mixture was heated for 7 hours at 80 ° C and the degree of conversion was periodically monitored by TLC or HPLC. After approximately 7 hours, HPLC indicated the presence of a 7-cyano compound. The mixture was then stirred overnight and allowed to cool to room temperature (about 22 ° C). To this mixture was added 200 ml of water, then 200 ml of methylene chloride and the resulting biphasic mixture was stirred, after which the mixture was left to separate phases. The aqueous layer was a gel. To this aqueous layer, in an unsuccessful attempt to destroy this gel, 100 ml of sodium bicarbonate solution was added. Then the aqueous layer was discarded.

, Aldrich). Фильтрат концентрировали досуха при пониженном давлении и при 45°С, с использованием устройства для отсасывания воды, и получали около 53,9 г сырого твердого продукта. Затем этот сырой твердый продукт растворяли в 50 мл метиленхлорида и обрабатывали 40 мл 4н соляной кислоты в делительной воронке и двухфазную смесь оставляли для отстаивания. Метиленхлоридный слой промывали 50 мл воды. Объединенные водные слои экстрагировали 50 мл метиленхлорида. Затем объединенные метиленхлоридные слои сушили сульфатом натрия и получали 45 г твердого вещества, которое представляло собой смесь 11α-гидроксиканренона и продукта 7α-циано-11α,17-дигидрокси-3-оксо-17α-прегн-4-ен-21-карбоновой кислоты, γ-лактона.The separated methylene chloride layer was washed with 100 ml of water and the resulting biphasic mixture was stirred. The mixture was then allowed to separate the phases and the separated methylene chloride layer was filtered through 200 g of silica gel (200-400 mesh sieve, 60

, Aldrich). The filtrate was concentrated to dryness under reduced pressure and at 45 ° C, using a device for suctioning water, and received about 53.9 g of crude solid product. Then this crude solid was dissolved in 50 ml of methylene chloride and treated with 40 ml of 4N hydrochloric acid in a separatory funnel and the two-phase mixture was left to settle. The methylene chloride layer was washed with 50 ml of water. The combined aqueous layers were extracted with 50 ml of methylene chloride. Then, the combined methylene chloride layers were dried with sodium sulfate to give 45 g of a solid, which was a mixture of 11α-hydroxycanrenone and the product 7α-cyano-11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-21-carboxylic acid, γ-lactone.

A sample of this product was analyzed by HPLC (column: 25 cm × 4.6 mm, 5 μm C ₁₈ LL Altima); solvent gradient: solvent A = water / trifluoroacetic acid = 99.9 / 0.1, solvent B = acetonitrile / trifluoroacetic acid = 99, 9 / 0.1, flow rate = 1.00 ml / min, gradient = 65: 30 (v / v) (A: B-first); 35:65 (v / v) (A: B - after 20 minutes); 10:90 (v / v) (A: B - after 25 minutes), detector on a diode array, which detected λ _max 238 nm.

The reaction mixture was analyzed by HPLC-NMR using the following conditions: HPLC column: Zorbax RX-C8 (25 cm × 4.6 mm, 5 microns) using a solvent gradient of 75% D ₂ O, 25% acetonitrile — 25% D ₂ O, 75% acetonitrile for 25 minutes at a flow rate of 1 ml / min;

¹ H NMR spectrum (obtained using WET solvent suppression) 5.84 (s, 1H), 4.01 (m, 1H), 3.2 (m, 1H), 2.9-1.4 (m, the full signal is not significant due to the suppression of acetonitrile), 0.93-0.86 (s, overlapping 3H, and t, 2H).

Example 20C. Preparation of 5β, 7α-dicyano-17-hydroxy-3-oxo-17α-pregnan-21-carboxylic acid, γ-lactone

102 g (0.3 mol) of 17-hydroxy-3-oxo-17α-pregna-4,6-diene-21-carboxylic acid, γ-lactone (canrenone) was suspended from 46.8 g (0.72 mol) of cyanide potassium, 78.6 ml (1.356 mol) of acetic acid and 600 ml of methanol in a three-liter three-necked round-bottom flask. To this mixture was added 64.8 ml (0.78 mol) of pyrrolidine, the combined suspension was heated under reflux (64 ° C) and kept for about 1.5 hours. Then the temperature of the suspension was reduced to 25 ° C-30 ° C for ten minutes using a cooling bath. 120 ml of concentrated hydrochloric acid was slowly added during cooling, during which precipitation of a tan solid was observed.

This mixture was stirred at 25 ° C-30 ° C for 1.5 hours, and then another 500 ml of water was added after 30 minutes. This mixture was cooled to 5 ° C in an ice bath and the pH was adjusted to 3 to 5.5 (pH was monitored using strips of indicator paper) by adding 100 ml of aqueous 9.5 M sodium hydroxide (0.95 mol). Excess cyanide was decomposed by adding household bleach. 25 ml (0.020 mol) was added to conduct a negative test with starch iodide. The cold mixture (10 ° C.) was filtered and the solids were washed with water until the washing reached a neutral pH (pH indicator paper). The solid was dried at 60 ° C. until a constant weight of 111.4 g was obtained.

The isolated solid was melted at 244-246 ° C on a Fisher Jones block. A methanol solution containing a solid did not exhibit absorption in the UV region of 210-240 nm. IR (CHCl ₃ ) cm ^-1 2222 (cyanide), 1775 (lactone), 1732 (3-keto). ¹ H-NMR (pyridin-d ₅ ) ppm 0.94 (s, 3H); 1.23 (s, 3H).

Example 21A

Scheme 1: Step 2: Preparation of 4'S (4'α), 7'α-hexadeca-hydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-trioxospiro- [furan -2 (3H), 17'β- [4,7] methano [17H] cyclopenta- [a] phenanthrene] -5'β (2'H) -carbonitrile

A 200 gallon enameled reactor (757 dm ³ ) was charged with 50 kg of enamine 2, approximately 445 L of 0.8 N diluted hydrochloric acid and 75 L of methanol. The mixture was heated for 5 hours to 80 ° C, and then cooled for 2 hours to 0 ° C. The solid product was filtered to obtain 36.5 kg of dry diketone product.

¹ H NMR (DMSO-d ₆ ): 4.53 (1H, d, J = 6), 3.74 (2H, m), 2.73 (1H, dd, J = 14.7), 2.65 -2.14 (8H, m), 2.05 (1H, t, J = 11), 1.98-1.71 (4H, m), 1.64 (1H, m), 1.55 (1H dd, J = 13.5), 1.45-1.20 (7H, m), 0.86 (3H, s).

Example 21B

Scheme 1: Stage 1 and 2: in situ preparation of 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5.20'-three -oxospiro [furan-2 (3H), 17'β (4,7) -methane [17H] cyclopenta [a] -phenanthrene] -5'β (2'H) -carbonitrile from 11α-hydroxycanrenone

100 g (280.54 mol) of 11-hydroxycanrenone obtained by the method described in Example 1 were loaded into a reactor equipped with a refrigerator, a mechanical stirrer, a heating jacket, an automatic regulator and a funnel, and then 300 ml of dimethylacetamide (Aldrich) was added. The mixture was stirred until 11-hydroxycanrenone was dissolved. To this mixture was added 31.5 ml of 50% sulfuric acid (Fisher), which contributed to raising the temperature of the resulting mixture to about 10 ° C-15 ° C. Then, a solution of sodium cyanide obtained by dissolving 31.18 g (617.20 mol) (Aldrich) of sodium cyanide in 54 ml of deionized water was added to a mixture of 11α-hydroxycanrenone for 2-3 minutes. After adding a solution of sodium cyanide, the temperature of the resulting mixture increased to about 20 ° C-25 ° C.

This mixture was heated to 80 ° C and maintained at this temperature for 2-3 hours. After HPLC analysis indicated that the conversion of 11α-hydroxycanrenone to enamine was substantially complete (conversion of more than 98%), the heat source was removed. Without isolating the enamine in the mixture, another 148 ml of 50% sulfuric acid was added to this mixture over 3-5 minutes. Then, 497 ml of deionized water was added to the mixture over 10 minutes.

This mixture was heated to 102 ° C and maintained at this temperature until approximately 500 g of distillate was removed from the mixture. During the reaction / distillation, 500 ml of deionized water was added to the mixture in four separate 125 ml portions. Each portion was added to the mixture after removal of an equivalent amount of distillate (approximately 125 ml). The reaction lasted for 2 hours. After HPLC analysis indicated that the hydrolysis of enamine to diketone was basically completed (conversion by more than 98%), the mixture was cooled to about 80 ° C for 20 minutes.

This mixture was filtered through a glass funnel. The reactor was washed with 1.2 L of deionized water to remove residual product. The solid on the filter was washed three times with approximately equal portions (about 0.4 L) of wash water. A 1 L solution of methanol and deionized water (1: 1, v / v) was obtained in the reactor, and the filtrate was washed with 500 ml of this solution. The filtrate was then washed twice with the remaining 500 ml of methanol / water. A vacuum was created in the funnel to dry the filtrate to a degree sufficient to transfer it. The filtrate was transferred to a drying oven, where it was dried in vacuo for 16 hours to obtain 84 g of a dry diketone product, 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β- dimethyl-3 ', 5,20'-trioxospiro [furan-2 (3H), 17'β (4,7) -methane- [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile . HPLC analysis showed 94% of the desired diketone.

Example 22

Scheme 1: Stage 3A: Method A: Preparation of methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

The 4-necked 5-liter round-bottom flask was equipped with a mechanical stirrer, a dropping funnel to equalize the pressure with a nitrogen inlet tube, a thermometer and a bubbler refrigerator. This bubbler was connected via a tigon tube system to two 2-liter traps, the first of which was empty and placed in a reaction vessel to prevent back-suction of material in the second trap (1 liter of concentrated sodium hydrochlorite solution). Diketone 3 (79.50 g; [weight was not adjusted for purity, which was 85%]) was added to a flask in 3 L of methanol. A 25% methanol solution of sodium methoxide (64.83 g) was poured into a funnel and was added dropwise, stirring in a nitrogen atmosphere, over 10 minutes. After complete addition, the orange-yellow reaction mixture was refluxed for 20 hours. After this period, 167 ml of 4N HCl was added dropwise to the still boiling reaction mixture through a dropping funnel (Note: HCN is released at this stage!). The reaction mixture, which was light in color, turned pale golden orange. Then the refrigerator was replaced with a head nozzle for removal and 1.5 L of methanol was removed by distillation, while 1.5 L of water was simultaneously added to the flask through a funnel in accordance with the rate of distillation. The reaction mixture was cooled to room temperature and was extracted twice with 2.25 L aliquots of methylene chloride. The combined extracts were washed successively with 750 ml aliquots of a cold saturated NaCl solution, 1N NaOH and again with a saturated NaCl solution. The organic layer was dried over sodium sulfate overnight, filtered and concentrated in vacuo to a volume of ~ 250 ml. Then, toluene (300 ml) was added and the remaining methylene chloride was evaporated under reduced pressure, during which time the product began to form on the flask walls as a white solid. The contents of the flask were cooled overnight and the solid was removed by filtration. This material was washed with 250 ml of toluene and then twice with 250 ml aliquots of ether and dried in a vacuum funnel, whereby 58.49 g of a white solid was obtained, which, as shown by HPLC, had a purity of 97.3%. After concentrating the mother liquor, another 6.76 g of product was obtained with a purity of 77.1%. The total yield, adjusted for purity, was 78%.

¹ H-NMR (CDCl ₃ ): 5.70 (1H, s), 4.08 (1H, s), 3.67 (3H, s), 2.9-1.6 (19H, m), 1 5-1.2 (5H, m); 1.03 (3H, s).

Example 23

Scheme 1: Step 3B: Conversion of methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone to methylhydro-17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregn- 4-en-7α, 21-dicarboxylate, γ-lactone

The 5-liter four-necked flask was equipped as described above, except that a trap system was not connected to the bubbler. Hydroxy ester in the amount of 138.70 g and then 1425 ml of methylene chloride was added to the flask with stirring under a nitrogen atmosphere. The reaction mixture was cooled to −5 ° C. using a salt / ice bath. Then methanesulfonyl chloride (51.15 g, 0.447 mol) was quickly added, after which triethylamine (54.37 g) in 225 ml of methylene chloride was slowly added dropwise. An addition that takes ~ 30 minutes was made so that the temperature of the reaction mixture never rose above about 5 ° C. After the addition, stirring was continued for 1 hour and the contents of the reactor were transferred to a 12-liter separatory funnel to which 2100 ml of methylene chloride was added. This solution was washed successively with 700 ml aliquots of cold 1N HCl, 1N NaOH and saturated NaCl solution (each). The water washes were combined and back extracted with 3500 ml of methylene chloride. All organic washes were combined in a 9 L vessel into which 500 g of neutral alumina with activity class II and 500 g of anhydrous sodium sulfate were added. The contents of this vessel were thoroughly mixed for 30 minutes and filtered. The filtrate was evaporated to dryness in vacuo to give a gummy yellow foam. This foam was dissolved in 350 ml of methylene chloride and 1800 ml of ether was added dropwise with stirring. The rate of addition was adjusted so that about half of the ether was added over 30 minutes. After adding about 750 ml, the product began to stand out as a crystalline solid. The remaining ether was added over 10 minutes. The solid was removed by filtration, and the filter cake was washed with 2 L of ether and dried in a vacuum oven at 50 ° C. overnight to give 144.61 g (88%) of an almost white solid, mp 149-150 ° C. The product thus obtained was usually 98-99% pure, as shown by HPLC (% area). In one cycle, a product was obtained having a melting point of 153-153, 5 ° C, with a purity of 99.5%, as determined by HPLC area.

¹ H-NMR (CDCl ₃ ): 5.76 (1H, s), 5.18 (1H, dt), 3.68 (3H, s), 3.06 (3H, s), 2.85 (1H , m), 2.75-1.6 (19H, m), 1.43 (3H, s), 1.07 (3H, s).

Example 24

Scheme 1: Stage 3C: Method A: Preparation of 7-methylhydro-17α-hydroxy-3-oxopregn-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone

A 1 L four-necked flask was equipped as described in the second example. Formic acid (250 ml) and acetic anhydride (62 ml) were added to this flask under stirring in a nitrogen atmosphere. Then potassium formate (6.17 g) was added and the reaction mixture was heated in an oil bath to an internal temperature of 40 ° C (this procedure was repeated at 70 ° C with best results) for 16 hours. After 16 hours, mesylate was added and the internal temperature was raised to 100 ° C. Heating and stirring were continued for another 2 hours, after which the solvent was removed in vacuo on a rotary evaporator. The residue was stirred with 500 ml of ice water for fifteen minutes, and then was extracted twice with 500 ml aliquots of ethyl acetate. The organic phases were combined and washed successively with 250 ml aliquots of a cold saturated sodium chloride solution (twice), a 1N sodium hydroxide solution and again saturated sodium chloride. Then the organic phase was dried with sodium sulfate, filtered and concentrated to dryness in vacuo, resulting in a yellowish-white foam, which was applied by spraying on a glass with a spatula. Analysis of 14.65 g of the obtained powder (% by area, HPLC) indicated that this powder was a mixture of 82.1% 7-methylhydro-17α-hydroxy-3-oxopregna-4.9 (11) -diene-7α, 21 β-dicarboxylate, γ-lactone; 7.4% 7-methylhydro-17α-hydroxy-3-oxopregna-4,11-diene-7α, 21-dicarboxylate, γ-lactone; and 5.7% 9α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, bis (γ-lactone).

¹ H-NMR (CDCl ₃ ): 5.74 (1H, s), 5.67 (1H, m), 3.61 (3H, s), 3.00 (1H, m), 2.84 (1H , ddd, J = 2.6, 15), 2.65-2.42 (6H, m), 2.3-2.12 (5H, m), 2.05-1.72 (4H, m) 1.55-1.45 (2H, m), 1.42 (3H, s), 0.97 (3H, s).

Example 25

Scheme 1: Stage 3C: Method B: Preparation of methylhydro-17α-hydroxy-3-oxopregn-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone

In a 5-liter four-necked flask, which was equipped as described above, 228.26 g of acetic acid and 41.37 g of sodium acetate were added while stirring under nitrogen. Using an oil bath, the mixture was heated to an internal temperature of 100 ° C. Mesylate (123.65 g) was then added and heating continued for another thirty minutes. At the end of this time period, heating was stopped and 200 ml of ice water was added. The temperature was lowered to 40 ° C and continued to stir for another 1 hour, after which the reaction mixture was slowly poured into 1.5 l of cold water in a 5-liter stirred flask. The product was isolated as a gummy oil. This oil was dissolved in 1 L of ethyl acetate and washed with 1 L of a cold saturated solution of sodium chloride, 1 N sodium hydroxide (each), and finally again with saturated sodium chloride. The organic phase was dried with sodium sulfate and filtered. The filtrate was concentrated to dryness in vacuo to give a foam which settled to form a gummy oil. This oil was triturated with ether for a period of time and it eventually cured. The resulting solid was filtered and washed with more ether to obtain 79.59 g of a yellow-white solid. This substance consisted of 70.4% of the target complex Δ ^9,11- ether 6; 12.3% of the complex Δ ^11,12- ether 8; 10.8% of 7-α, 9-α-lactone 9 and 5.7% of unreacted compound 5.

Example 26

Scheme 1: Stage 3D: Method A: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

The 4-necked 500 ml jacketed reactor was equipped with a mechanical stirrer, a fridge / bubbler, a thermometer and a dropping funnel with a nitrogen supply tube. While stirring in a nitrogen atmosphere, 8.32 g of crude ester in 83 ml of methylene chloride were charged to this reactor. After that, 4.02 g of dibasic potassium phosphate was added, followed by 12 ml of trichloroacetonitrile. Water was passed through the jacket of the reactor for external cooling, and the reaction mixture was cooled to 8 ° C. 36 ml of 30% hydrogen peroxide was added to the dropping funnel over 10 minutes. After complete addition, the reaction mixture, initially having a pale yellow color, became almost colorless. During the addition, the reaction mixture was maintained at 9 ± 1 ° C and stirred overnight (23 hours total). Methylene chloride (150 ml) was added to the reaction mixture, and all contents were added to ~ 250 ml of ice water. This mixture was extracted three times with 150 ml aliquots of methylene chloride. The combined methylene chloride extracts were washed with 400 ml of cold 3% sodium sulfite solution to decompose any residual peroxide. After that, 330 ml of cold washing with 1N sodium hydroxide, 400 ml of cold washing with 1N hydrochloric acid, and finally washing with 400 ml of saturated saline were washed. The organic phase was dried with magnesium sulfate, filtered, and the filter cake was washed with 80 ml of methylene chloride. The solvent was removed in vacuo to give 9.10 g of the crude product as a pale yellow solid. This material was recrystallized from ~ 25 ml of 2-butanone to give 5.52 g of almost white crystals. After final recrystallization from acetone (~ 50 ml), 3.16 g of long needle crystals were obtained, mp. 241-243 ° C.

¹ H-NMR (CDCl ₃ ): 5.92 (1H, s), 3.67 (3H, s), 3.13 (1H, d, J = 5), 2.89 (1H, m), 2 81-2.69 (15H, m), 1.72 (1H, dd, J = 5.15), 1.52-1.22 (5H, m), 1.04 (3H, s).

Example 27

Scheme 1: Stage 3: Option 1: Conversion of 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tri-oxospiro [furan-2 (3H), 17'β (4,7) -methane [17H] cyclopenta [a] -phenanthrene] -5'β (2'H) -carbonitrile in methylhydro-9,11α-epoxy-17α- hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

Diketone (20 g) was charged into a clean and dried reactor, and then 820 ml of MeOH and 17.6 ml of a 25% NaOMe / MeOH solution were added. The reaction mixture was heated under reflux (~ 67 ° C) for 16-20 hours. The product was quenched with 40 ml of 4N HCl. The solvent was removed at atmospheric pressure by distillation. Then, 100 ml of toluene was added, and residual methanol was removed by azeotropic distillation with toluene. After concentration, the crude hydroxy ester 4 was dissolved in 206 ml of methylene chloride and cooled to 0 ° C. Thereafter, methanesulfonyl chloride (5 ml) was added, and then 10.8 ml of triethylamine was slowly added. This product was stirred for 45 minutes. The solvent was removed by vacuum distillation to give crude mesylate 5.

5.93 g of potassium formate, 240 ml of formic acid, and then 118 ml of acetic anhydride were added to a separate dried reactor. The mixture was heated to 70 ° C. for 4 hours.

A mixture containing formic acid was added to concentrated solution 5, prepared as described above. This mixture was heated to 95-105 ° C for 2 hours. The mixture containing the product was cooled to 50 ° C and the volatile components were removed by vacuum distillation at 50 ° C. The product was partitioned between 275 ml of ethyl acetate and 275 ml of water. The aqueous layer was back-extracted with 137 ml of ethyl acetate, washed with 240 ml of a cold 1N sodium hydroxide solution, and then with 120 ml of saturated NaCl. After phase separation, the organic layer was concentrated by vacuum distillation to give the crude ester.

The product was dissolved in 180 ml of methylene chloride and cooled to 0-15 ° C. After that, 8.68 g of dipotassium biphosphate was added, followed by 2.9 ml of trichloroacetonitrile. To the resulting mixture was added 78 ml of a solution of 30% hydrogen peroxide over 3 minutes. The reaction mixture was stirred for 6-24 hours at 0-15 ° C. After the reaction, the biphasic mixture was allowed to separate phases. The organic layer was washed with 126 ml of a 3% sodium sulfate solution, 126 ml of a 0.5N sodium hydroxide solution, 126 ml of 1N hydrochloric acid, and 126 ml of a 10% saturated saline solution. The product was dried with anhydrous sodium sulfate or filtered on celite, after which the solvent methylene chloride was removed by distillation at atmospheric pressure. The product was crystallized twice from methyl ethyl ketone to give 7.2 g of epoxymexrenone.

Example 28

Scheme 1: Stage 3: Option 2: Conversion of 1'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tri-oxospiro [furan-2 (3H), 17'β (4,7) -methane [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile in methylhydro-9,11α-epoxy-17α-hydroxy -3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone, without isolation of the intermediate.

A 4-necked round-bottom 5-L flask was equipped with a mechanical stirrer, a dropping funnel with a nitrogen supply tube, a thermometer and a refrigerator with a bubbler attached to a scrubber with sodium hypochlorite. Diketone (83.20 g) was added to a flask in 3.05 L of methanol. 67.85 g of a 25% (w / w) solution of sodium methoxide in methanol was charged into a dropping funnel. Under stirring in a nitrogen atmosphere, methoxide was added dropwise to the flask over 15 minutes. The result was a dark orange / yellow suspension. The reaction mixture was heated under reflux for 20 hours, and 175 ml of 4N hydrochloric acid was added dropwise, while continuing heating under reflux. (Caution, HCN is released during this reaction!). The reflux condenser was changed to an exhaust head and 1.6 L of methanol was removed by distillation, adding 1.6 L of an aqueous 10% sodium chloride solution dropwise through a funnel in accordance with the distillation rate. The reaction mixture was cooled to room temperature and was extracted twice with 2.25 L aliquots of methylene chloride. The combined extracts were washed with cold 750 ml aliquots of 1N sodium hydroxide and saturated sodium chloride solution. The organic layer was dried by azeotropic distillation of methanol under pressure in one atmosphere to a final volume of 1 l (0.5% of the total product was taken for analysis).

The concentrated organic solution (hydroxy ester) was again added to the original reaction flask equipped as previously described but without a trap for HCN. This flask was cooled to 0 ° C. and 30.7 g of methanesulfonyl chloride was added while stirring under nitrogen. 32.65 g of triethylamine was added dropwise into the dropping funnel over 15 minutes, maintaining the temperature at 5 ° C. Stirring was continued for 2 hours, while the reaction mixture was warmed to room temperature. Then a column was prepared containing 250 g of Dowex 50 W × 8-100 acid-ion exchange resin, and before that it was washed with 250 ml of water, 250 ml of methanol and 500 ml of methylene chloride. The reaction mixture was removed from this column and collected. Then a fresh column was prepared and the above procedure was repeated. After that, a third column was prepared containing 250 g of a basic ion exchange resin Dowex 1 × 8-200 and pretreated as the acid resin column described above. The reaction mixture was removed from this column and collected. Then, a fourth column of ion exchange resin was prepared and the reaction mixture was again removed and collected. Then, two 250 ml portions of methylene chloride washes were passed through each column, and each run required ~ 10 minutes. Solvent washes were combined with the reaction mixture and concentrated in vacuo to a volume of ~ 500 ml, and 2% of this volume was removed for quantification. The residue was then concentrated to a final volume of 150 ml (crude mesylate solution).

960 ml of formic acid, 472 ml of acetic anhydride and 23.70 g of potassium formate were added to the initial 5-liter reaction apparatus. This mixture was heated with stirring in a nitrogen atmosphere to 70 ° C for 16 hours. The temperature was then raised to 100 ° C. and a crude mesylate solution was added over a dropping funnel over 30 minutes. As methylene chloride was distilled from the reaction mixture, the temperature was lowered to 85 ° C. After all the substance was removed, the temperature rose again to 100 ° C and was maintained for 2.5 hours. The reaction mixture was cooled to 40 ° C, and formic acid was removed under pressure to achieve a minimum stirring volume (~ 150 ml). The residue was cooled to room temperature and 375 ml of methylene chloride was added. The diluted residue was washed with cold 1-liter portions of a saturated solution of sodium chloride, 1N sodium carbonate and again a solution of sodium chloride. The organic phase was dried with magnesium sulfate (150 g) and filtered to give a dark tan solution (crude solution of the ester).

The 4-necked 1-liter jacketed reactor was equipped with a mechanical stirrer, a refrigerator / bubbler, a thermometer and a dropping funnel with a nitrogen supply tube. A crude solution of the ester (approximately 60 g) in 600 ml of methylene chloride was charged into the reactor with stirring under nitrogen. After this was added 24.0 g of dibasic potassium phosphate, and then 87 ml of trichloroacetonitrile. Water was supplied through the jacket of the reactor for external cooling, and the reaction mixture was cooled to 10 ° C. This mixture was added over a 30 minute period to a dropping funnel with 147 ml of 30% hydrogen peroxide. After completion of the addition, the reaction mixture, initially colored in dark tan, turned a light yellow color. During the entire period of addition, the reaction mixture was maintained at 10 ± 1 ° C and stirring was continued overnight (a total of 23 hours). The phases were separated and the aqueous portion was extracted twice with 120 ml portions of methylene chloride. Then, the combined organic phases were washed with 210 ml of a 3% sodium sulfite solution. This procedure was repeated once more, after which the organic and aqueous parts showed a negative reaction to peroxide, as evidenced by indicator paper with starch / iodide. The organic phase was washed successively with 210 ml aliquots of cold 1N sodium hydroxide, 1N hydrochloric acid and, finally, two washes with saturated saline. The organic phase was dried by azeotropic distillation to a volume of ~ 100 ml, then fresh solvent (250 ml) was added and subjected to azeotropic distillation similarly to ~ 100 ml, and the remaining solvent was removed in vacuo to obtain 57.05 g of crude product as a gummy yellow foam. Then a portion (51.01 g) of the product was dried to a constant weight of 44.3 g and quantified by HPLC. Analysis indicated 27.1% epoxymexrenone.

Example 29. Obtaining 3-ethoxy-11α-hydroxy-androsta-3,5-diene-17-one from 11α-hydroxyandrostenedione

11α-hydroxyandrostenedione (429.5 g) and toluenesulfonic acid hydrate (7.1) were introduced into the reaction flask under a nitrogen atmosphere. Ethanol (2.58 L) was added to the reactor and the resulting solution was cooled to 5 ° C. Triethylorthoformate (334.5 g) was added to this solution over 15 minutes at a temperature of 0 ° C to 15 ° C. After completion of the addition of triethyl formate, the reaction mixture was heated to 40 ° C and allowed to react at this temperature for 2 hours, after which the temperature was raised to reflux temperature, and the reaction was continued under reflux for another 3 hours. The reaction mixture was cooled in vacuo and the solvent was removed in vacuo to give 3-ethoxy-11α-hydroxyandrosta-3,5-dien-17-one.

Example 30. Obtaining enamine from 11α-hydroxycanrenone

Scheme 1: Step 1: Method B: Preparation of 5'R (5'α), 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'α, 13'α-dimethyl-3 ', 5-dioxospiro [furan-2 (3H), 17'α (5'H) - [7,4] metheno [4H [cyclopenta [a] -phenanthrene] -5'-carbonitrile.

Sodium cyanide (1.72 g) was placed in a 25 ml 3-necked flask equipped with a mechanical stirrer. Then water (2.1 ml) was added and the mixture was stirred while heating until the solids dissolved. After that, dimethylformamide (15 ml) was added, followed by 11α-hydroxycanrenone (5.0 g). A mixture of water (0.4 ml) and sulfuric acid (1.49 g) was added to the resulting mixture. This mixture was heated to 85 ° C for 2.5 hours, during which HPLC analysis showed complete conversion to the product. The reaction mixture was cooled to room temperature. Then sulfuric acid (0.83 g) was added and the mixture was stirred for half an hour. The reaction mixture was added to 60 ml of water, cooled in an ice bath. The flask was washed with 3 ml of DMF and 5 ml of water. The suspension was stirred for 40 minutes and filtered. The filter cake was washed twice with 40 ml of water and dried in a vacuum oven at 60 ° C overnight to give 11α-hydroxyenamine, i.e. 5'R (5'α), - 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'α, 13'α-dimethyl-3 ', 5-dioxospiro [furan-2 (3H), - 17'α (5'H) - [7.4] metheno [4H] cyclopenta [a] phenanthrene] -5'-carbonitrile (4.9 g).

Example 31. The conversion of 11α-hydroxycanrenone to diketone in one tank

Sodium cyanide (1.03 g) was added to a 50 ml 3-necked flask equipped with a mechanical stirrer. After adding water (1.26 ml), the flask was slightly heated to dissolve the solid. Then dimethylacetamide [or dimethylformamide] (9 ml) was added, followed by 11α-hydroxycanrenone (3.0 g). A mixture of sulfuric acid (0.47 ml) and water (0.25 ml) was added to the reaction flask with stirring. The resulting mixture was heated to 95 ° C for 2 hours. HPLC analysis indicated the completion of the reaction. Then sulfuric acid (0.27 ml) was added and the mixture was stirred for 30 minutes. After that, additional water (25 ml) and sulfuric acid (0.90 ml) were added and the reaction mixture was stirred for 16 hours. Then the mixture was cooled in an ice bath to 5-10 ° C. The solid was isolated by filtration through a sintered glass filter, followed by a double wash with water (20 ml). Solid diketone, i.e. 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5-20'-trioxospiro [furan-2 (3H), - 17'β (4.7) methano [17H] cyclopenta [a] phenanthrene] -5′β (2′H) -carbonitrile was dried in a vacuum oven to give 3.0 g of a solid.

Example 32A-1.

Scheme 1: Stage 3A: Method B: Preparation of methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

A suspension of 5.0 g of diketone obtained by the method described in Example 31 in methanol (100 ml) was heated under reflux and a 25% solution of potassium methoxide in methanol (5.8 ml) was added over 1 minute. The mixture became homogeneous. After 15 minutes, a precipitate appeared. The mixture was heated under reflux and after about 4 hours, it again became homogeneous. Reflux was continued for a full 23.5 hours and 4.0 n HCl (10 ml) was added. All 60 ml of a solution of hydrogen cyanide in methanol was removed by distillation. Water (57 ml) was added to the residue obtained by distillation over 15 minutes. During the addition of water, the temperature of the solution was raised to 81.5 ° C and another 4 ml of a solution of hydrogen cyanide / methanol was removed by distillation. After the addition of water was completed, the mixture became cloudy and the heat source was removed. The resulting mixture was stirred for 3.5 hours and the product crystallized slowly. The suspension was filtered and the collected solid was washed with water, dried in a stream of air on a funnel, and then dried at 92 ° C (26 in.Hg) for 16 hours to obtain 2.98 g of a whitish solid. This solid was 91.4% hydroxy ester, i.e. methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-carboxylate, γ-lactone (by weight). The yield was 56.1%.

Example 32A-2. Preparation of methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

The diketone (40 g) obtained by the method described in Example 31 was loaded into a cleaned, dry 1-liter jacketed reactor equipped with a bottom drain, a refrigerator, an RTD sensor, and a fraction collector. Methanol (800 ml) was charged into this reactor and the mixture was stirred. The resulting suspension was heated to about 60-65 ° C. and a 25% potassium methoxide solution (27.8 ml) was added. The mixture became homogeneous.

The resulting mixture was heated under reflux. After about 1.5 hours under reflux, another 16.7 ml of a 25% potassium methoxide solution was added to the mixture under reflux. The mixture was maintained under reflux for another 6 hours. The conversion of diketone to hydroxy ester was analyzed by HPLC. After HPLC analysis showed a ratio of diketone to hydroxy ester of less than about 10%, 77 ml of 4M HCl was added to the mixture over reflux for about 15 minutes (hydrochloric acid could be replaced with a comparable amount of 1.5M-3M sulfuric acid )

The mixture was then distilled and about 520 ml of methanol / HCN distillate was collected and discarded. The concentrated mixture was cooled to about 65 ° C. About 520 ml of water was added to the mixture over 90 minutes and the temperature was maintained at about 65 ° C during the addition. The mixture was gradually cooled to about 15 ° C for approximately four hours, and then stirred and maintained at a temperature of about 15 ° C for another two hours. The mixture was filtered and the filtered product was washed twice with about 200 ml of water (each time). The filtered product was dried in vacuo (90 ° C, 25 mmHg). Received approximately 25-27 g of a whitish solid, containing mainly methylhydro-11α, 17α-dihydroxy-3-oxo-pregn-4-en-7α, 21-dicarboxylate, γ-lactone.

Example 32B-1 Preparation of 7-methylhydro-5β-cyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone

4.1 g of diketone obtained by the method described in Example 31, 75 ml of methanol and 1 ml of a 1N methanol solution of sodium hydroxide were charged into the reaction flask. The suspension was stirred at room temperature. After a few minutes, a homogeneous solution was obtained, and after about 20 minutes a precipitate was observed. Stirring was continued for another 70 minutes at room temperature. After this time, the solid precipitate was filtered and washed with methanol. The solid precipitate was dried in a steam chamber, whereby 3.6 g of 7-methylhydro-5β-cyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone were obtained.

¹ H-NMR (CDCl ₃ ) ppm 0.95 (s, 3H), 1.4 (s, 3H), 3.03 (d, 1H, J = 15), 3.69 (s, 3H), 4.1 (m, 1H).

¹³ C-NMR (CDCl ₃ ) ppm 14.6, 19.8, 22.6, 29.0, 31.0, 33.9, 35.17, 35.20, 36.3, 37.7, 38.0, 38.9, 40, 8, 42.8, 43.1, 45.3, 45.7, 47.5, 52.0, 68.0, 95.0, 121.6, 174.5, 176.4, 207.0.

Example 32B-2 Preparation of 7-methylhydro-5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone

To 2.0 g (4.88 mol) of 9,11-epoxydiketone of formula 21 suspended in 30 ml of anhydrous methanol was added 0.34 ml (2.4 mmol) of triethylamine. The suspension was heated under reflux and after 4.5 hours the presence of starting material was not observed, as indicated by HPLC (Zorbax SB-C8, 150 × 4.6 mm, 2 ml / min, linear gradient 35:65 A: B-45: 55 A: B for 15 minutes, A = acetonitrile / methanol 1: 1, B = water / 0.1% trifluoroacetic acid, detection at 210 nm). The mixture was left to cool and kept at about 26 ° C for about 16 hours. The resulting suspension was filtered to give 1.3 g of 7-methylhydro-5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone, as a white solid . The filtrate was concentrated to dryness on a rotary evaporator, and the residue was triturated with 3-5 ml of methanol. After filtration, another 260 mg of 7-methylhydro-5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone was obtained. The yield was 74.3%.

¹ H NMR (400 MHz, deuterochloroform) δ: 1.00 (s, 3H), 1.45 (m, 1H), 1.50 (s, 3H), 1.65 (m, 2H), 2.10 (m, 2H), 2.15-2.65 (m, 8H), 2.80 (m, 1H), 2.96 (m, 1H), 3.12 (d, J = 13, 1H), 3.35 (d, J = 7, 1H); 3.67 (s, 3H).

Example 32C. Preparation of 5β-cyano-11-α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone

6.8 g of diketone (obtained by the method described in Example 31), 68 ml of acetonitrile, 6.0 g of sodium acetate and 60 ml of water were charged into the reaction flask. The mixture was heated and stirred under reflux. After 1.5 hours, the mixture became almost homogeneous. After 3 hours, 100 ml of water was added as 50 ml of acetonitrile was distilled. The mixture was cooled and the precipitated solid (1.7 g) was removed by filtration. The filtrate (pH = 5.5) was treated with hydrochloric acid to lower the pH to about 4.5 and a solid precipitated. The solid was isolated, washed with water and dried, resulting in 4.5 g of 5β-cyano-11-α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone.

¹ H-NMR (DMSO) ppm 0.8 (s, H), 1.28 (s, H), 3.82 (m, 1H).

¹³ C-NMR (DMSO) ppm 14.5, 19.5, 22.0, 28.6, 30.2, 33.0, 34.1, 34.4, 36.0, 37.5, 37.7, 38.5, 42, 4, 42.6, 45.08, 45.14, 47.6, 94.6, 122.3, 176.08, 176.24, 207.5.

Example 33

Scheme 1: Stage 3A: Method C: Preparation of methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The diketone (30 g) obtained by the method described in Example 31 was charged into a purified and dried 3-necked reaction flask equipped with a thermometer, a Dean-Stark trap, and a mechanical stirrer. Methanol (24 ml) was loaded into the reactor at room temperature (22 ° C) and the resulting suspension was stirred for 5 minutes. The reactor was loaded with 25% of the mass. a solution of sodium methoxide in methanol (52.8 ml) and the resulting mixture was stirred at room temperature for 10 minutes, during which the reaction mixture turned into a light brown transparent solution and a slight temperature increase was observed (by 2-3 ° C). The rate of addition was adjusted to prevent a temperature increase in the tank above 30 ° C. Then the mixture was heated under reflux (about 67 ° C) and continued to heat under reflux for 16 hours. A sample was then taken and analyzed by conversion HPLC. The reaction mixture was continued to reflux until the residual diketone level exceeded 3% of the diketon charge. During reflux, 4N HCl (120 ml) was charged into the reaction tank, resulting in HCN formation, which was quenched in a scrubber.

After the reaction, 90-95% methanol solvent was distilled off from the reaction mixture at atmospheric pressure. The temperature in the head during the distillation process ranged from 67 to 75 ° C, and the distillate that contained HCN was treated with caustic soda and bleach, and then disposed of. After methanol was removed, the reaction mixture was cooled to room temperature, and as the mixture was cooled to between 40-45 ° C, a solid product began to precipitate. An aqueous solution, optionally containing 5% of the mass. sodium bicarbonate (1200 ml) at 25 ° C was loaded into a cooled suspension, and then the resulting mixture was cooled to 0 ° C for about 1 hour. Treatment with potassium bicarbonate is effective to remove residual unreacted diketone from the reaction mixture. The suspension was stirred at 0 ° C for 2 hours to complete the precipitation and crystallization, after which the solid product, methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone, was isolated by filtration and the filter cake was washed with water (100 ml). The product was dried at 80-90 ° C in vacuo under a pressure of 26 "mercury (inch mmHg) to constant weight. The water content after drying was less than 0.25% by mass. The adjusted molar yield was in the range of 77-80 % of the mass.

Example 34

Scheme 1: Stage 3A: Method D: Preparation of methylhydro-11α, 17α-dihydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The diketone obtained by the method described in Example 31 (1 equiv.) Was reacted with sodium methoxide (4.8 equiv.) In a methanol solvent in the presence of zinc iodide (1 equiv.). The product was processed either by the method of extraction described in this application or by the method without extraction, in which there were no stages of extraction with methylene chloride, washing with saturated saline and caustic soda, and drying with sodium sulfate. In addition, in a method not involving extraction, toluene was replaced with 5% of the mass. sodium bicarbonate solution. Methylhydro-11α, 17α-dihydroxy-3-oxo-pregn-4-en-7α, 21-dicarboxylate, γ-lactone were isolated as the product.

Example 35

Scheme 1: Stage 3C: Method C: Preparation of methylhydro-17α-hydroxy-3-oxopregn-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone

The hydroxy ester obtained as described in Example 34 (1.97 g) was combined with tetrahydrofuran (20 ml) and the resulting mixture was cooled (-70 ° C). Sulfuryl chloride (0.8 ml) was then added and the mixture was stirred for 30 minutes, after which imidazole (1.3 g) was added. The reaction mixture was warmed to room temperature and stirred for another 2 hours. The mixture was then diluted with methylene chloride and extracted with water. The organic layer was concentrated to obtain a crude product of methylhydro-17α-hydroxy-3-oxopregn-4.9 (11) -diene-7α, 21-dicarboxylate, γ-lactone (1.97 g). A small sample of the crude product was analyzed by HPLC. Analysis showed that the ratio of 9.11-olefin: 11.12-olefin: 7.9-lactone was 75.5: 7.2: 17.3. If the reaction was carried out at 0 ° C or otherwise, as described above, then this reaction gave a product in which the ratio of 9.11-olefin: 11.12-olefin: 7.9-lactone was 77.6: 6.7: 15 , 7. This procedure was combined into one step of introducing the leaving group and removing it for the subsequent administration of the 9.11-olefin structure of the ester, i.e. reaction with sulfuryl chloride leads to the replacement of the 11α-hydroxy group of the hydroxy ester of formula V with a halide followed by dehydrohalogenation with the formation of a Δ ^9.11 structure. Thus, the formation of an ester proceeds efficiently without the use of a strong acid (such as formic acid) or a desiccant, such as acetic anhydride. The stage of reflux heating carried out in an alternative method in which carbon monoxide is formed is also excluded.

Example 36A

Scheme 1: Stage 3C: Method D: Preparation of methylhydro-17α-hydroxy-3-oxopregn-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

The hydroxy ester (20 g) obtained as described in Example 34 and methylene chloride (400 ml) were added to a cleaned dry three-necked round-bottom flask equipped with a mechanical stirrer, dropping funnel and thermocouple. The resulting mixture was stirred at room temperature until complete formation of the solution. The solution was cooled to 5 ° C. using an ice bath. Methanesulfonyl chloride (5 ml) was added to a CH ₂ Cl ₂ solution containing a hydroxy ester, and then triethylamine (10.8 ml) was slowly added immediately dropwise. The rate of addition was adjusted so that the reaction temperature did not exceed 5 ° C. This reaction was very exothermic, and therefore needed to be cooled. The reaction mixture was stirred for 1 h at about 5 ° C. Upon completion of the reaction (HPLC and TLC analyzes), the mixture was concentrated at about 0 ° C under a pressure of 26 inches Hg. until a thick suspension forms. The resulting suspension was diluted with CH ₂ Cl ₂ (160 ml) and the mixture was concentrated at about 0 ° C under a pressure of 26 inches of mercury. with obtaining a concentrate. It was found that the purity of the concentrate (mesylate product of formula IV, where R ³ = H and -A-A- and -B-B- represent -CH ₂ -CH ₂ -, i.e. the conversion of methylhydro-11α, 17α-dihydroxy-3 -oxo-pregn-4-en-7α, 21-dicarboxylate, γ-lactone to methylhydro-17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregn-4-en-7α, 21-di-carboxylate, γ- lactone) is 82% (% of area, HPLC). This material was used in the next reaction without isolation.

Potassium formate (4.7 g), formic acid (16 ml) and acetic anhydride (8 ml, 0.084 mol) were added to a clean dry reactor equipped with a mechanical stirrer, a refrigerator, a thermocouple and a heating jacket. The resulting solution was heated to 70 ° C and stirred for about 4-8 hours. The addition of acetic anhydride led to an exothermic reaction and the formation of gas (CO), and therefore, the rate of addition was adjusted accordingly to control temperature and gas formation (pressure). The reaction time to obtain the active eliminating reagent depended on the amount of water present in the reaction (formic acid and potassium formate contained about 3-5% water, each). The elimination reaction is sensitive to the amount of water present; that is, if this amount is> 0.1% water (KF), then the level of the 7.9-lactone impurity can be increased. This by-product is difficult to remove from the final product. If KF is <0.1% water, then the active eliminating agent was added to the mesylate concentrate (0.070 mol) obtained in the previous step. The resulting solution was heated to 95 ° C, and the volatile material was distilled off and collected in a Dean-Stark trap. When the evolution of volatile material ceased, the Dean-Stark trap was replaced with a refrigerator and the reaction mixture was heated for another 1 hour at 95 ° C. After this time (TLC and HPLC analysis; <0.1% of the starting material), the contents were cooled to 50 ° C and distillation started in vacuo (26 inch Hg / 50 ° C). The mixture was concentrated to obtain a thick suspension, and then cooled to room temperature. The resulting suspension was diluted with ethyl acetate (137 ml) and the solution was stirred for 15 minutes, and then diluted with water (137 ml). The layers were separated and the aqueous layer was again extracted with ethyl acetate (70 ml). The combined ethyl acetate solution was washed once with saturated saline (120 ml) and twice with ice-cold 1N NaOH solution (120 ml each). The pH of the aqueous layer was measured, and if the pH of the spent washing was <8, then the organic layer was washed again. If the pH of the spent washing was> 8, then the ethyl acetate layer was washed once with brine (120 ml) and concentrated to dryness on a rotary evaporator using a 50 ° C water bath. The result was 92 g of the ester, a solid product, i.e. methylhydro-17α-hydroxy-3-oxopregn-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone (yield 77% mol).

Example 36B Preparation of methylhydro-17α-hydroxy-3-oxo-pregna-4.9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

25 g (53.12 mmol) of methylhydro-11α, 17α-dihydroxy-3-oxopregna-4-en-7α, 21-dicarboxylate was added to a clean dry three-necked round-bottomed 250 ml flask equipped with a mechanical stirrer, dropping funnel and thermocouple , γ-lactone obtained as described in Example 34, and then 150 ml of methylene chloride (Burdick & Johnson). The resulting mixture was stirred at room temperature until a clear suspension was obtained. The solution was cooled to -5 ° C in an ice bath. Methanesulfonyl chloride (7.92 g, 69.06 mmol) (Aldrich) was added to a methylene chloride solution containing a hydroxy ester (Aldrich), and then triethylamine (7.53 g) (Aldrich) was slowly added dropwise immediately. The rate of addition was adjusted so that the reaction temperature did not exceed 0 ° C. This reaction was very exothermic; and therefore she needed cooling. The addition time was 35 minutes. The reaction mixture was stirred at about 0 ° C for another 45 minutes. Upon completion of the reaction (when less than 1% hydroxyether remained, as indicated by HPLC and TLC analysis), the mixture was concentrated by evaporation of approximately 110-125 ml of methylene chloride solvent at atmospheric pressure. During evaporation, the reaction temperature reached approximately 40-45 ° C. If after 45 minutes the reaction has not been completed, then another 0.1 equivalent of methanesulfonyl chloride and another 0.1 equivalent of triethylamine can be loaded into the reactor, followed by an assessment of the reaction to completion. The resulting mixture contained the crude product methylhydro-17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone. This product was used in the subsequent reaction without isolation.

Anhydrous sodium acetate (8.7 r) (Mallinkrodt), glacial acetic acid (42.5 ml) (Fisher) and acetic anhydride were added to a second 250 ml cleaned dry reactor equipped with a mechanical stirrer, a refrigerator, a thermocouple and a heating jacket. (0.5 ml) (Fisher). The resulting solution was heated to 90 ° C and stirred for about 30 minutes. Since the addition of acetic anhydride is accompanied by an increase in temperature, the addition was carried out at such a rate that it corrected both temperature and pressure. Acetic anhydride was added to reduce the water content in the solution to an acceptable level (less than about 0.1%). If KF indicated <0.1% water, then the acetic acid solution was transferred to the mesylate concentrate obtained as reported in the first section of this example. After transfer, the temperature of the resulting mixture was from about 55 ° C to 60 ° C. Gas production was reduced using acetic acid and sodium acetate instead of formic acid and potassium formate, as described in Example 36A.

The resulting mixture was heated to 135 ° C and maintained at the same temperature for about 60-90 minutes until the evolution of volatiles ceased. Volatiles distilled from the mixture were collected in a Dean-Stark trap. After completion of the reaction (TLC and HPLC analysis; <0.1% of the starting material), the heat source was removed. When the temperature of the mixture reached 80 ° C, 150 ml of water was slowly added to this mixture over 60-90 minutes. Upon completion of the addition of water, the mixture was cooled to a temperature of from about 35 ° C to 45 ° C and a suspension began to form. Then the mixture was cooled to 15 ° C and maintained at this temperature from about 30 to 60 minutes.

The resulting mixture was filtered through a glass funnel.

The filtrate was washed with 100 ml of water. Then the filtrate was washed a second time with another 100 ml of water. The obtained filtrate was dried at 70 ° C in vacuo to obtain 25.0 g of a dry ester product, methylhydro-17α-hydroxy-3-oxopregn-4.9 (11) -diene-7α, 21-dicarboxylate, γ-lactone. HPLC analysis indicated the presence of 70% of the desired 9.11 olefin, 15% 11.12 olefin and 5% 7.0 lactone.

This method gave a good result (compared to similar methods), namely: (i) reduces the volume of solvents, (ii) reduces the number of individual work steps required to produce the ester from the hydroxy ester, (iii) eliminates the need for washing, (iv) by isolating the final product, it is possible to replace the extraction by precipitation with water; and (v) provides reliable elimination, which was previously associated with the formation of mixed anhydride and gas evolution using formic acid instead of acetic acid.

Example 37A

Scheme 1: Stage 3C: Method E: Preparation of methylhydro-17α-hydroxy-3-oxopregn-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

A hydroxy ester (100 g, 0.22 mol) was added to a three-necked round-bottomed 2 L flask equipped with a mechanical stirrer, dropping funnel and thermocouple. The circulating cooling bath was equipped with an automatic temperature controller. Before starting the reaction, the flask was drained due to the sensitivity of methanesulfonyl chloride to water.

Methylene chloride (1 L) was charged into the flask and the hydroxy ester was dissolved therein by stirring. The solution was cooled to 0 ° C and methanesulfonyl chloride (25 ml; 0.32 mol) was charged through a dropping funnel into the flask. Triethylamine (50 ml, 0.59 mol) was charged into the reaction flask through a dropping funnel, and the funnel was washed with additional methylene chloride (34 ml). The addition of triethylamine was highly exothermic. The addition time was about 10 minutes with stirring and cooling. The loaded mixture was cooled to 0 ° C and kept at this temperature, while stirring for another 45 minutes, during which the head region of the reaction flask was purged with nitrogen. Then, a sample of the reaction mixture was analyzed by thin layer chromatography and high resolution liquid chromatography to monitor the progress of the reaction. After this, the mixture was stirred at 0 ° C for another 30 min and again evaluated for completion of the reaction. The analysis showed that at this stage the reaction was basically completed; the methylene chloride solvent was evaporated at 0 ° C in vacuo at 26 "Hg. Analysis of the distillate by gas chromatography indicated the presence of methanesulfonyl chloride and triethylamine. Then methylene chloride (800 ml) was charged into the reaction flask and the resulting mixture was stirred for 5 minutes at a temperature of the range of 0-15 ° C. After this, the solvent was again evaporated at 0-5 ° C under a pressure of 26 "Hg and received a mesylate of the formula IV, where R ³ represents H; -A-A- and -B-B-are —CH ₂ —CH ₂ -, and R ¹ is methoxycarbonyl. The purity of the product was about 90-95% (area).

To obtain an eliminating reagent, potassium formate (23.5 g; 0.28 mol), formic acid (80 ml) and acetic anhydride (40 ml) were mixed in a separate dried reactor. Formic acid and acetic anhydride were fed into the reactor by a pump, and during the addition of acetic anhydride, the temperature was kept at no more than 40 ° C. The elimination reagent mixture was heated to 70 ° C. to drain water from the reaction system. This reaction was continued until the water content was lower than 0.3 mass%, as measured by Karl Fischer analysis. The elimination reagent solution was then transferred to a reactor containing a concentrated crude mesylate solution prepared as described above. The resulting mixture was heated to a maximum temperature of 95 ° C and the volatile distillate was collected until the distillate was no longer formed. Distillation ceased at about 90 ° C. After completion of the distillation, the reaction mixture was stirred at 95 ° C. for another 2 hours, and the completion of the reaction was monitored by thin layer chromatography. When the reaction was completed, the reactor was cooled to 50 ° C, and formic acid and the solvent were removed from the reaction mixture under vacuum at 26 "Hg at 50 ° C. The concentrate was cooled to room temperature, and then ethyl acetate (688 ml) was introduced. and a mixture of ethyl acetate and concentrate was stirred for 15 minutes, a 12% saline solution (688 ml) was added at this stage to facilitate removal of water-soluble impurities from the organic phase, the phases were left to settle for 20 minutes, and the aqueous layer was transferred to another vessel into which uploaded optional e amount of ethyl acetate (350 ml). This back extraction of the aqueous layer was carried out for 30 minutes, after which the phases were allowed to precipitate and the ethyl acetate layers were combined. Saturated sodium chloride solution (600 ml) was added to the combined ethyl acetate layers and stirring was carried out for 30 minutes, the phases were then allowed to precipitate, the aqueous layer was removed, then an additional washing with sodium chloride (600 ml) was carried out, and the organic phase was separated from the second spent washing solution. After that, the organic phase was washed with 1N sodium hydroxide (600 ml) with stirring for 30 minutes. The phases were precipitated for 30 minutes to remove the aqueous layer. After evaluating the pH of the aqueous layer, it was found to be> 7. Additional washing with saturated sodium chloride (600 ml) was carried out for 15 minutes. Finally, the organic phase was concentrated in vacuo at 26 mm Hg. at 50 ° C and the product was isolated by filtration. After drying, the final product was a brown foamy solid. This substance was further dried at 45 ° C. under reduced pressure for 24 hours to obtain 95.4 g of an ether product, methylhydro-17α-hydroxy-3-oxopregna-4.9 (11) -diene-7α, 21- dicarboxylate, γ-lactone, which was analyzed to be 68.8%. The molar yield was 74.4%, which was adjusted both for the initial hydroxy ester and the final ester.

Example 37B Preparation of 7-methylhydro-17-methyl-3-oxo-18-norpregna-4.9 (11), - 13-trien-7α, 21-dicarboxylate

5.5 g of mesylate obtained by the method described in Example 23, 55 ml of 94.3% formic acid and 1.38 g of potassium formate were loaded into the reaction flask. The mixture was heated and stirred under reflux (104 ° C) for two hours. After these two hours, formic acid was distilled off under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10% potassium carbonate (50 ml). The isolated water was yellow in color. Ethyl acetate was washed with 5% sodium hydroxide (50 ml). The aqueous parts were combined and acidified with dilute hydrochloric acid, and the insoluble matter was extracted with ethyl acetate. Ethyl acetate was evaporated to dryness under reduced pressure to obtain 1.0 g of a residue, 7-methylhydro-17-methyl-3-oxo-18-norpregna-4.9 (11), 13-triene-7α, 21-dicarboxylate.

¹ H-NMR (CDCl ₃ ) m: d: 1.5 (s, 3H), 1.4 (s, 3H), 3.53 (s, 3H), 5.72 (m, 1H).

¹³ C-NMR (CDCl ₃ ) ppm: 25.1 and 25.4 (18 CH ₃ and 19 CH ₃ ), 40.9 (10 C), 48.5 (17 C), 51.4 ( OCH ₃ ), 118.4 (11 CH), 125.4 (4 CH), 132.4 (9 C), 138.5 and 139.7 (13 C and 14 C), 168.2 (5 C) , 172.4 (7 CO), 179.6 (22 CO), 198.9 (3 CO).

Example 37C Preparation of 7-methylhydro-5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ-lactone.

5.5 g of mesylate, prepared as described in Example 23, 55 ml of 94.3% formic acid and 1.38 g of potassium formate were charged into the reaction flask. The mixture was heated and stirred under reflux (104 ° C) for two hours. After these two hours, formic acid was distilled off under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10% potassium carbonate (50 ml). The isolated water was yellow in color. Ethyl acetate was washed with 5% sodium hydroxide (50 ml). Ethyl acetate was evaporated to dryness under reduced pressure to obtain 3.7 g of a residue. A portion (3.4 g) of the residue was chromatographed over 267 g of Merck silica gel (40-63 microns). The product was isolated by elution with ethyl acetate and toluene (37:63) (v / v). After drying of this product, 0.0698 g of residue, 7-methylhydro-5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ-lactone, was obtained.

¹ H-NMR (CDCl ₃ ) ppm: 1.03 (s, 3H), 1.22 (s, 3H), 3.70 (s, 3H), 5.60 (d, 1H, J = 10), 5.98 (d, 1H, J = 10).

MICC ^-1 : 2229 (CN), 1768 (lactone), 1710 (ester).

Example 37D Isolation of 9α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, bis (γ-lactone)

9α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, bis (γ-lactone) is a by-product of the elimination of 11-mesylate. A pure sample was isolated from the reaction mixture of Example 37A using preparative liquid chromatography, and then using reverse phase preparative HPLC. Thus, 73 g of the residue was chromatographed on 2.41 kg of Merck silica gel (40-63 microns) according to the gradient elution scheme with ethyl acetate and toluene (20:80, 30:70, 40:60, 60:40 v / v). An enriched mixture (10.5 g) of enamine and 7.9-lactone was obtained in fractions in a ratio of 60:40. The purification progress was monitored by TLC on plates with EMF, eluting (60:40 v / v) with ethyl acetate and toluene, and visualized with sulfuric acid, SWUV. Then, a portion (10.4 g) of the mixture was purified by reverse phase HPLC on Kromasil C8 (7 μm) with a mobile phase: water milliQ and acetonitrile, 30:70 v / v. 7.9-lactone (2.27 g) in the form of crystals was isolated from the mobile phase.

MICC ^-1 : 1762 (7.9-lactone and 17-lactone), 1677, 1622 (3-keto-Δ ^4.5 ).

¹ H-NMR (CDCl ₃ ) ppm: 1.00 (s, 3H), 1.4 (s, 3H), 2.05 (d, 1H), 2.78 (d, 1H), 5 87 (s, 1H).

¹³ C-NMR (CDCl ₃ ) ppm: 13.2, 19.0, 22.2, 23.2, 26.8, 28.8, 29.5, 30.8, 33.1, 34 , 4, 35.1, 42.5, 43.6, 43.9, 45.0, 45.3, 89.9, 94.7, 129.1, 161.5, 176.0, 176.4 , 196.9.

Calculated: C 71.85 and H 7.34; Found: C, 71.68; and H, 7.30.

Example 37E Isolation of 7-methylhydro-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ-lactone

The compound 7-methylhydro-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ-lactone was isolated after repeated preparative liquid chromatography of the reaction mixture obtained after the 11- elimination reaction mesyloxy groups (Example 24). It is part of an accumulation of less polar impurities, as evaluated by TLC on EMF plates using an elution system with ethyl acetate and methylene chloride (30:70 v / v) and visualized with sulfuric acid, SWUV. Basically, these less polar impurities were isolated from crude enamine by preparative liquid chromatography. In particular, 9.6 g of crude enamine solution was chromatographed on 534 g of Merck silica gel (40-63 microns) using a gradient elution scheme with ethyl acetate and toluene (20:80, 30:70, 40:60, 60:40 v / v) . Less polar impurities were concentrated in fractions in a ratio of 30:70. In the same way, 12.5 g of a pool of less polar impurities was collected. Then, this product was chromatographed over 550 g of Merck silica gel (40-63 μm) using a gradient elution scheme with ethyl acetate and methylene chloride (5:95, 10:90, 20:80, 30:70 v / v). The enriched portion of 20:80 fractions gave 1.2 g of residue. Additional chromatography of 1.2 g of residue on 53 g of Merck silica gel (40-63 μm) using a gradient of acetone and methylene chloride (3:97, 6:94, 10:90, 15:85 vol./about.) Gave 0.27 g of 7-methylhydro-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-en-7α, 21-dicarboxylate, γ-lactone from the enriched portion of the 10:90 fractions.

MS M + 425 calculated for C ₂₅ H ₃₁ NO ₅ (425.52).

MIC 2222 cm ^-1 (nitrile), 1767 cm ^-1 (lactone), 1727 cm ^-1 (ester and 3-ketone).

¹ H-NMR (CDCl ₃ ) ppm 0.92 (s, 3H), 1.47 (s, 3H), 2.95 (m, 1H), 3.65 (s, 3H), 5.90 (m, 1H).

¹³ C-NMR (CDCl ₃ ) ppm 14.0 (18 CH ₃ ), 23.5 (15 CH ₂ ), 27.0 (19 CH ₃ ) 37.8, 38.5 and 40.9 (7.8 and 14 CH), 52.0 ( OCH ₃ ), 95.0 (17 C), 121.5 (23 CN), 123.5 (11 CH), 135.3 (9 C), 174.2 and 176.2 (22 and 24 CO), 206 (3 CO).

Example 37F Preparation of 7-methylhydro-17-hydroxy-3-oxo-11α- (2,2,2-trifluoro-1-oxoethoxy) -17α-pregn-4-en-7α, 21-dicarboxylate

The hydroxy-ester (2.0 g, 4.8 mmol) obtained by the method described in Example 34 was added to 40 ml of methylene chloride in a clean dry 3-necked round bottom flask equipped with a mechanical stirrer. Then, triethylamine (0.61 g, 6.10 mmol) and trifluoroacetic anhydride (1.47 g, 7.0 mmol) were added to the solution. This mixture was stirred overnight at room temperature.

Then the mixture was diluted with another 40 ml of methylene chloride. After that, the mixture was washed successively with 40 ml of water, 40 ml of 1N HCl and 40 ml of a 1N NaOH solution. The resulting solution was then dried with magnesium sulfate and concentrated to dryness, whereby 3.2 g of a light brown solid, 7-methylhydro-17-hydroxy-3-oxo-11α- (2,2,2-trifluoro-1-oxoethoxy) were obtained ) -17α-pregn-4-en-7α, 21-dicarboxylate.

The residue was analyzed and purified by chromatography. Conditions for HPLC: column - Waters Symmetry C18 (150 mm × 4.6 mm internal diameter; particle size 5 microns); column temperature - indoor; mobile phase - acetonitrile / water, 30/70 by volume; flow rate - 1.0 ml / min; injection volume - 20 microliters; sample concentration - 1.0 mg / ml; UV detection at 210 nm; pressure - 1500 psi (105.5 kg / cm ² ); and the run time is 45 minutes. Conditions for TLC: adsorbent - silica gel Merck 60 F ₂₅₄ ; solvent system - ethyl acetate / toluene, 65/35 by volume; visualization technique - shortwave; and the amount of application is 100 micrograms.

Example 37G Preparation of 7-methylhydro-11α- (acetyloxy) -17-hydroxy-3-oxo-17α-pregn-4-en-7,21-dicarboxylate, γ-lactone

The hydroxy-ester (2.86 g, 6.87 mmol) obtained by the method described in Example 34 was added to 40 ml of methylene chloride in a clean dry 3-necked round bottom flask equipped with a mechanical stirrer. Then, triethylamine (1.39 g, 13.7 mmol), dimethylaminopyridine (0.08 g, 0.6 mmol) and acetic anhydride (1.05 g, 10.3 mmol) were added to the solution. This mixture was stirred overnight at room temperature.

Then the mixture was diluted with another 150 ml of ethyl acetate and 25 ml of water. After that, the ethyl acetate solution was washed with 25 ml of citric acid solution. The resulting solution was then dried with magnesium sulfate and concentrated to dryness, whereby 3.33 g of a light brown solid, 7-methylhydro-11α- (acetyloxy) -17-hydroxy-3-oxo-17α-pregn-4-ene, were obtained 7,21-dicarboxylate, γ-lactone.

The residue was then analyzed and purified by chromatography. Conditions for HPLC: column - Waters Symmetry C18 (150 mm × 4.6 mm internal diameter; particle size 5 microns); column temperature - room; mobile phase - acetonitrile / water 30/70 by volume; flow rate - 1.0 ml / min; injection volume - 20 microliters; sample concentration - 1.0 mg / ml; UV detection at 210 nm; pressure - 1500 psi (105.5 kg / cm ² ); and the run time is 45 minutes. Conditions for TLC: Merck silica gel adsorbent 60 F ₂₅₄ ; solvent system - methylene chloride / methanol 95/5 by volume; visualization technique - shortwave; and the amount of application is 100 micrograms.

Example 37H.

Scheme 1: Stage 3C: Method F: Preparation of 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone

Potassium formate (1.5 g, 0.018 mol), formic acid (60 ml, 1.6 mul) and acetic anhydride (29.5 ml, 0.31 mol) were added to a clean, dry 250 ml reactor equipped with a mechanical stirrer, fridge, thermocouple and a casing for heating. The solution was then stirred for 4 hours at 70 ° C. and cooled to room temperature, whereby an elimination reagent was obtained, which was used to convert the mesylate of formula IV into the product of this example.

The pre-obtained elimination reagent TFA / TFA anhydride was added to 70.0 g (0.142 mol) of mesylate obtained by the method described in Example 23. The resulting mixture was heated to 95-105 ° C for 2.5 hours, while the degree of conversion was periodically monitored using TLC or HPLC. The resulting residue was cooled to 50 ° C, diluted with ice water (1.4 L) and stirred for 1 hour. The mixture was left to stand overnight at room temperature. The layers were separated and the aqueous phase was again extracted with ethyl acetate (75 ml). Then, the ethyl acetate solution was washed successively with a mixture of water / brine (70 ml), another mixture of water / brine (60 ml), 1N sodium hydroxide (60 ml) and a third mixture of water / brine (60 ml). The strength of the saturated saline solution was 12% of the mass. Then, the ethyl acetate solution was dried with sodium sulfate, filtered and concentrated to dryness on a rotary evaporator, whereby 4.5 g of a mixture of the desired product and an unknown impurity were obtained. The impurity / product ratio determined by HPLC area was about 50/15, respectively. The predominant product of this reaction was an impurity that was identified as 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4.9 (11) -diene-7.21-dicarboxylate, γ-lactone.

The mixture was purified by column chromatography to give 1.9 g of analytically pure 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4.9 (11) diene-7α, 21-dicarboxylate, γ-lactone.

The residue was again analyzed and purified by chromatography. Conditions for HPLC: column - Waters Symmetry C18 (150 mm × 4.6 mm internal diameter; particle size 5 microns); column temperature - indoor; mobile phase - acetonitrile / water 30/70 by volume; flow rate - 1.0 ml / min; injection volume - 20 microliters; sample concentration - 1.0 mg / ml; UV detection at 210 nm; pressure - 1500 psi (105.5 kg / cm ² ); and the run time is 45 minutes. Conditions for TLC: Merck silica gel adsorbent 60 F ₂₅₄ ; solvent system - chloroform / methyl tert-butyl ether / isopropanol, 70/28/2 by volume; visualization technique - 50% vol. aqueous H ₂ SO ₄ / LWUV and 50% vol. H ₂ SO ₄ / phosphomolybdic acid; and the amount of application is 100 micrograms.

Example 37I Preparation of 7-methylhydro-17-hydroxy-3,11-dioxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

The Jones reagent was prepared by dissolving 6.7 g of chromic anhydride (CrO ₃ ) in 6 ml of concentrated sulfuric acid, and this mixture was carefully diluted with distilled water to a volume of 50 ml. One ml of this reagent was enough to oxidize 1 mmol of secondary alcohol to give a ketone.

The hydroxy ester (10.0 g, 24.0 mmol) obtained by the method described in Example 34 was dissolved / suspended in 1200 ml of acetone. 8.992 ml of Jones reagent was added to this mixture, and the combined mixture was stirred for 10 minutes. An aliquot of the reaction mixture after it was treated with water and extracted with methylene chloride was analyzed by HPLC (column: Beckman Ultrasphere ODS C18, 4.6 mm × 250 mm, 5 microns; solvent gradient: acetonitrile / water = 1 / 99-100 / 0 for 20 minutes at a flow rate of 1.5 ml / min; detector: UV 210 nm). The completion of the reaction was indicated by the absence of any significant amount of starting material in the reaction mixture. The retention time for the starting material (hydroxy-ester) was 13.37 minutes, and for the ketone product it was 14.56 minutes.

The reaction mixture was treated by adding 200 ml of water and 300 ml of methylene chloride. The organic layer was separated from the aqueous layer and washed again with 200 ml of water. The organic layer was separated from the aqueous layer and dried with magnesium sulfate. The solvent was evaporated to give 9.52 g of a whitish solid (95.6% crude yield) 7-methylhydro-17-hydroxy-3,11-dioxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ- lactone.

The structure was confirmed based on the mass spectrum (m / e 414). H-NMR (DMF-d7) and C-NMR (DMF-d7). In H-NMR, the characteristic 11-H peak (4.51 ppm, doublet, J = 5.8 Hz), found in the hydroxyether ester, was absent. In C-NMR, a peak was detected at 208.97 ppm, which, presumably, corresponds to 11-keto carbon.

C-NMR (400 MHz, DMF-d7) 208.97 (11-keto), 197.70 (3-keto), 176.00 (22-lactone), 173.34 (7-COOMe), 167.21 (C5), 125.33 (C4), 93.63 (C17) and other peaks in the region from 15 to 57 ppm.

Example 37J Preparation of dimethyl-11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

A solution of 3.5 g (8.4 mmol) of the hydroxy ester prepared as described in Example 34 in 42 ml of methanol was mixed with 4 ml of methanol 4n potassium hydroxide (8 mmol). The suspension was stirred overnight at room temperature and heated under reflux for one hour. Methanol was evaporated in vacuo and the residue was mixed with 50 ml of ethyl acetate. Ethyl acetate was evaporated in vacuo, and the residue was hydrolyzed with 50 ml of ethyl acetate. The dried solid was combined with 50 ml of acetone and 2 ml of methyl iodide (32.1 mmol). The mixture was stirred at room temperature for 18 hours. During this time, most of the solids dissolved. The mixture was filtered and the filtrate was evaporated to dryness in vacuo. The residue was hydrolyzed with ethyl acetate, after which the solids were removed by filtration, and the solvent was removed by vacuum distillation. The residue was defined as a mixture of 78:22 (v / v) dimethyl-11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone and the hydroxy ester starting material as was determined using ¹ H-NMR. This mixture was suitable for use as an HPLC marker without further purification.

¹ H-NMR (CDCl ₃ ) showed the following characteristic spectrum: ppm 0.93 (s, 3H), 1.37 (s, 3H), 3.64 (s, 3H), 3.69 (s, 3H).

Example 37K. Preparation of 11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

To 11.86 g (28.5 mmol) of the hydroxy ester obtained by the method described in Example 34, 50 ml of methanol and 20 ml of 2.5 M NaOH were added. The suspension was heated under reflux. After 25 minutes, a portion of the starting ester, which, as shown by HPLC, remained unreacted (Zorbax SB-C8 150 × 4.6 mm, 2 ml / min, linear gradient 35:65 A: B - 45:55 A: B for 15 minutes, A = acetonitrile / methanol 1: 1, B = water / 0.1% trifluoroacetic acid, detection at 210 nm) and 10 ml of 10 M NaOH was added. After 1.5 hours, as shown by HPLC, only trace amounts of unreacted ester remained. The reaction mixture was left for approximately 64 hours at about 25 ° C.

The mixture was diluted with 100 ml of water, and then strongly acidified by adding 20 ml of concentrated HCl. The resulting gummy precipitate was stirred until this precipitate became a suspension. The solids were isolated by filtration, resuspended in methanol and filtered, resulting in 3.75 g of a brown solid. This substance was dissolved in 8 ml of hot DMF and the mixture was diluted with 40 ml of methanol. The acid was crystallized and isolated by filtration, whereby 1.7 g of a flocculent white solid, 11α, 17-dihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone was obtained.

¹ H-NMR (400 MHz, deuterodimethyl sulfoxide) δ 0.80 (s, 3H), 1.25 (s, 3H), 1.2-2.7 (m, 20H), 3.8 (br s, 1H), 4.45 (m, 1H), 5.50 (s, 1H). No carboxyl proton was observed due to the presence of a HOD peak at 3.4 ppm.

Example 38

Scheme 1: Stage 3C: Method G: Preparation of 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone

The procedure of Example 37A was repeated except that, due to the treatment of the reaction solution with an ion exchange resin, basic alumina, or basic silica, many washing steps were not performed. The conditions for treatment with basic alumina or basic silica are described in Table 38. It was determined that each of these treatments was effective for removing impurities without multiple washing steps of Example 44, resulting in 7-methylhydro-17-hydroxy-3-oxo -17α-pregna-4.9 (11) -diene-7,21-dicarboxylate, γ-lactone.

Table 38. Factor Selected quantity Experiment goal Key Results Basic alumina 2 g / 125 g of product The reaction mixture basic aluminum oxide treatment to remove Et ₃ ^N. HCl salts and to remove 1N NaOH and 1N HCl washes The yield was 93% Basic silica 2 g / 125 g of product Treatment of the reaction mixture basic silica which is cheaper to remove Et ₃ ^N. HCl salts and to remove 1N NaOH and 1N HCl washes The yield was 95%

Example 39

Scheme 1: Stage 3C: Method H: Preparation of 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone

Potassium formate (4 g) and trifluoroacetic acid (42.5 ml) were mixed in a 100 ml reactor. Then, trifluoroacetic anhydride (9.5 ml) was added to the mixture at a controlled rate, keeping the temperature below 30 ° C during the addition. Then, the solution was heated to 30 ° C. for 30 minutes and cooled to room temperature, whereby an elimination reagent was obtained, which was used to convert the mesylate of formula IV into an ester of formula II.

The preformed elimination reagent TFA / TFA anhydride was added to a solution of the mesylate of formula IV obtained previously by the method described in Example 37A. The resulting mixture was heated to 40 ° C for 4.5 hours, while the degree of conversion was periodically monitored by TLC or HPLC. Upon completion of the reaction, the mixture was transferred to a 1-necked flask and concentrated to dryness under reduced pressure and at room temperature (22 ° C). Ethyl acetate (137 ml) was added to the mixture, as a result of which the solid phase material was completely dissolved, and then a mixture of water / saturated saline (137 ml) was added and the resulting two-phase mixture was stirred for 10 minutes. Then the mixture was left for 20 minutes to separate the phases. The strength of the saturated saline solution was 24% of the mass. The aqueous phase was contacted with additional ethyl acetate (68 ml) and the thus obtained two-phase mixture was stirred for 10 minutes, after which it was left for 15 minutes to separate the phases. The ethyl acetate layers after two extraction were combined and washed with 24% of the mass. saturated saline (120 ml), another aliquot of 24% of the mass. saturated saline (60 ml), 1N sodium hydroxide solution (150 ml) and another portion of saturated saline (60 ml). After each addition of the aqueous phase, the mixture was stirred for 10 minutes and left for 15 minutes to separate the phases. The remaining solution was concentrated to dryness under reduced pressure at 45 ° C. using a water suction device. The solid product (8.09 g) was analyzed by HPLC, and it was found that this product included 83.4% (area) of the 7-methyl-hydro-17-hydroxy-3-oxo-17α-pregnane ester 4.9 (11) -diene-7,21-dicarboxylate, γ-lactone; 2.45% (area) of 11,12-olefin; 1.5% 7.9-lactone; and 1.1% unreacted mesylate.

Example 40

Scheme 1: Stage 3C: Method I: Preparation of 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone

The mesylate having the structure obtained in Example 23 (1.0 g), isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) were placed in a 50 ml flask and heated to 90 ° C with stirring. After 5 hours, the mixture was cooled to 25 ° C and concentrated in vacuo at 10 mm Hg. The residue was dissolved in CH ₂ Cl ₂ (20 ml) and washed with 5% aqueous NaHCO ₃ . The CH ₂ Cl ₂ layer was concentrated in vacuo to give 1.47 g of a tan oil. This product was recrystallized from CH ₂ Cl ₂ / Et ₂ O to give 0.50 g of enol acetate of formula IV (Z).

This product was added to a mixture of sodium acetate (0.12 g) and acetic acid (2.0 ml), which was previously heated to 100 ° C. with stirring. After 60 minutes, the mixture was cooled to 25 ° C and diluted with CH ₂ Cl ₂ (20 ml). The solution was washed with water (20 ml) and dried with MgSO ₄ . The drying agent was removed by filtration and the filtrate was concentrated in vacuo to give 0.4 g of the desired 9.11-olefin, 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4.9 (11) -diene -7,21-dicarboxylate, γ-lactone. The crude product contained less than 2% impurities of 7.9-lactone.

Example 41. Thermal elimination of mesylate in DMSO

Scheme 1: Stage 3C: Method J: Preparation of 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone

A mixture of 2 g of mesylate and 5 ml of DMSO in a flask was heated at 80 ° C for 22.4 hours. HPLC analysis of the reaction mixture did not detect the starting material. Water (10 ml) was added to the reaction mixture, and the precipitate was extracted three times with methylene chloride. The combined methylene chloride layers were washed with water, dried with magnesium sulfate and concentrated to give the ester, 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone .

Example 42

Scheme 1: Stage 3D: Method B: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

In a pear-shaped 50 ml flask, an ester of formula IIA (1.07 g of an analyzed 74.4% of an ester), trichloroacetamide (0.32 g) and dipotassium bisphosphate (0.70 g) were mixed as a solid, while stirring with methylene chloride (15.0 ml). Then, hydrogen peroxide (30 wt%; 5.0 ml) was added with a pipette over 1 minute. The resulting mixture was stirred for 6 hours at room temperature, during which HPLC analysis showed that the ratio of epoxymexrenone to the ester in the reaction mixture was approximately 1: 1. An additional amount of trichloroacetamide (0.32 g) was added to the reaction mixture and the reaction was continued, stirring for another 8 hours, after which it was found that the residual amount of ester was reduced to 10%. An additional amount of trichloroacetamide (0.08 g) was then added and the reaction mixture was left overnight, after which only 5% of unreacted ester with respect to epoxymexrenone remained in the mixture.

Example 43

Scheme 1: Stage 3D: Method C: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

An ester of formula IIA (5.4 g, according to a quantitative analysis of 74.4% of the ester) was added to a 100 ml reactor. Trichloroacetamide (4.9 g) and dipotassium bisphosphate (3.9 g) were added to this complex ester, both in solid form, and then methylene chloride (50 ml) was added. The mixture was cooled to 15 ° C. and 30% hydrogen peroxide (25 g) was added over 10 minutes. The reaction mixture was left until it was heated to 20 ° C, and stirred at this temperature for 6 hours, after which an HPLC analysis was carried out for the degree of conversion. It was found that the amount of remaining ether was less than 1% of the mass.

This reaction mixture was added to water (100 ml) and the mixture was left to separate phases, after which the methylene chloride layer was removed. Sodium hydroxide (0.5 N, 50 ml) was added to the methylene chloride layer. After 20 minutes, the mixture was allowed to separate the phases, and then HCl (0.5 N; 50 ml) was added to the methylene chloride layer, after which the mixture was left to separate the phases and the organic phase was washed with brine (50 ml). The methylene chloride layer was dried with magnesium sulfate and the solvent was removed. The result was a white solid (5.7 g). The aqueous layer of sodium hydroxide was acidified and extracted, and the extract was treated to obtain another 0.2 g of product. The yield of epoxymexrenone was 90.2%.

Example 44

Scheme 1: Stage 3D: Method D: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The ester of formula IIA was converted to epoxymexrenone by the method described in Example 43, except for the following changes: the initial charge contained the ester (5.4 g, quantification: 74.4% of the ester), trichloroacetamide (3.3 g) and dipotassium bisphosphate (3.5 g). Then added hydrogen peroxide (12.5 ml). The reaction was carried out overnight at 20 ° C, after which, as shown by HPLC, 90% conversion of the ester to epoxymexrenone was observed. Then another 3.3 g of trichloroacetamide and 30% hydrogen peroxide (5.0 ml) were added and the reaction was carried out for another 6 hours, after which the amount of residual ester was only 2% based on the calculation of the loading of the ester. After treatment as described in Example 43, 5.71 g of epoxymexrenone was obtained.

Example 45

Scheme 1: Stage 3D: Method E: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The ester of formula IIA was converted to epoxymexrenone by the method described in Example 43. In the reaction of this example, the loading of the complex ether was 5.4 g (quantitative analysis: 74.4% of the complex ether), the loading of trichloroacetamide was 4.9 g, the loading of hydrogen peroxide was 25 g, and the loading of dipotassium phosphate was 3.5 g. The reaction was carried out for 18 hours at 20 ° C. The amount of residual ester was less than 2%. After treatment, 5.71 g of epoxymexrenone was obtained.

Example 45

Scheme 1: Stage 3D: Method F: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The ester of formula IIA was converted to epoxymexrenone by the method described in Example 43, except that the reaction temperature of this example was 28 ° C. The reactor charge contained an ester (2.7 g), trichloroacetamide (2.5 g) and dipotassium bisphosphate (1.7 g), hydrogen peroxide (17.0 g) and methylene chloride (50 ml). After 4 hours, the amount of residual ester was only 2% based on the calculation of the loading of the ester. After treatment as described in Example 43, 3.0 g of epoxymexrenone was obtained.

Example 47-1.

Scheme 1: Stage 3D: Method G: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The ester of formula IIA (40 g, quantitative analysis: 68.4% of the ester) was loaded into a 1000 ml jacketed reactor and dissolved in 175 ml of methylene chloride. The solution was stirred as trichloroacetamide (22.3 g) and dipotassium bisphosphate (6.0 g) were added as solids. This mixture was stirred at 400 rpm and the temperature was adjusted to 27 ° C. using a constant temperature bath to regulate the circulation of the liquid through the reactor jacket. Then, hydrogen peroxide (72.8 ml, assay: 30%) was added over 3-5 minutes. After adding hydrogen peroxide, the mixture was stirred at 400 rpm and at 27 ° C. HPLC analysis indicated that the reaction was complete in 5 hours (99%). After six hours, 72.8 ml of water was added. Aqueous hydrogen peroxide was separated and once subjected to back extraction with 50 ml of methylene chloride. The combined methylene chloride was washed with 6% sodium sulfite (62.3 ml) to decompose any remaining peroxide. Removal of methylene chloride was initiated by distillation at atmospheric pressure and placing in vacuum. Thus, a yellowish residue was obtained (48.7 g, 55.4% analysis). This corresponded to a yield of 94.8% obtained by analysis based on the molar yield.

A portion (47.8 g) of the residue was combined with 498 ml of ethanol 3A (95% ethanol denatured with 5% methanol). The resulting mixture was refluxed and 249 ml of distillate was removed at atmospheric pressure. The mixture was cooled to 25 ° C and filtered. Washing with ethanol 3A (53 ml) was used to facilitate transfer. The dried solid was 27.6 g (analysis: 87.0%), which corresponded to a yield of 91%. A portion of the solid (27.0 g) was dissolved in 292 ml of methyl ethyl ketone under reflux. The hot solution was filtered through a layer of Solkafloc (pulverized powder) with another 48.6 ml of methyl ethyl ketone, used to facilitate transfer. 146 ml of methyl ethyl ketone was removed by atmospheric distillation. The solution was cooled to 50 ° C. and stirred for one hour as the product crystallized. After one hour, the mixture was cooled to 25 ° C. Stirring was continued for one hour and the solid was filtered with 48.6 ml of methyl ethyl ketone, used as a wash. The solid was dried to a constant weight of 20.5 g, which amounted to a yield of 87.2% after recrystallization. The reaction yield and the isolated ethanol, methyl ethyl ketone were combined to give a total yield of 75%.

The methyl ethyl ketone mother liquor was suitable for re-cycling with the addition of methylene chloride solution from the subsequent reaction. A mixture of the combined methylene chloride and methyl ethyl ketone was evaporated to dryness by distillation at atmospheric pressure and in vacuum. The residue was combined with 19 volumes of ethanol 3A, based on the content of epoxymexrenone. One half of the solvent was removed by atmospheric distillation. After cooling to 25 ° C., the solid was filtered and dried. The dry solid was dissolved in 12 volumes of methyl ethyl ketone under reflux. The hot solution was filtered through a Solka Floc pad with 2 volumes of methyl ethyl ketone added as a wash. The filtrate was concentrated by distillation of 6 volumes of methyl ethyl ketone at atmospheric pressure. This solution was cooled to 50 ° C. and stirred for one hour as the product crystallized. After one hour, the mixture was cooled to 25 ° C. Stirring was continued for another hour and the solid was filtered with 2 volumes of methyl ethyl ketone, used as a wash. The solid product was dried until constant weight was obtained. The introduction of the mother liquor of methyl ethyl ketone increased the overall yield to 80-85%.

This method has proven to be particularly suitable for large-scale production because it helps maximize productivity and minimize flushing and waste volumes.

Example 47A

Scheme 1: Stage 3D: Method H: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone

The ester of formula IIA (17 g, quantitative analysis: 72% of the ester) was dissolved in methylene chloride (150 ml), after which trichloroacetamide (14.9 g) was added with slow stirring. The temperature of the mixture was brought to 25 ° C and a solution of dipotassium bisphosphate (10.6 g) in water (10.6 ml) was added to the solution of the ether substrate with stirring at a speed of 400 rpm. Then, hydrogen peroxide (30% wt. Solution; 69.4 ml) was added to the substrate / phosphate / trichloroacetamide mixture over 3-5 minutes. No exothermic reaction or oxygen evolution was observed. The reaction mixture thus obtained was stirred at 400 rpm for 18.5 hours at 25 ° C. No oxygen evolution was observed during the reaction, but analysis of the consumption of hydrogen peroxide showed that a certain amount of oxygen was formed during the reaction. The reaction mixture was diluted with water (69.4 ml) and the mixture was stirred at about 250 rpm for 15 minutes. During this reaction, temperature control was not necessary and the reaction was carried out mainly at room temperature (any temperature in the range of 5-25 ° C. is suitable). The aqueous and organic layers were allowed to separate and the lower methylene chloride layer was removed.

The aqueous layer was back extracted with methylene chloride (69.4 ml) for 15 minutes, stirring at 250 rpm. These layers were allowed to separate and the lower methylene chloride layer was removed. The aqueous layer (177 g; pH = 7) was analyzed for hydrogen peroxide. The result (12.2%) of the analysis indicated that 0.0434 mol of hydrogen peroxide was absorbed in the 0.0307 mol of olefin reaction. Excessive absorption of hydrogen peroxide is an indicator of the formation of oxygen in this reaction. The back extraction of a small volume of methylene chloride was a sufficient guarantee of the absence of loss of epoxymexrenone in the aqueous layer. This result coincides with the result obtained using a second extraction with a large amount of methylene chloride, in which only trichloroacetamide was isolated.

The combined methylene chloride solutions obtained from the above extracts were combined and washed with 3% of the mass. sodium sulfate solution (122 ml) for at least 15 minutes with stirring at a speed of about 250 rpm. At the end of the mixing period, a negative test was obtained using starch iodide (KI paper; no color was observed; in a positive test, a purple color indicated the presence of peroxide).

The aqueous and organic layers were allowed to separate, and the lower methylene chloride layer was removed. The aqueous layer (pH = 6) was discarded. It should be noted that the addition of a solution of sodium sulfite can cause a slight increase in temperature, and therefore this addition should be carried out under temperature control.

The methylene chloride phase was washed with 0.5 N sodium hydroxide (61 ml) for 45 minutes at a stirring speed of about 250 rpm and at a temperature in the range of 15-25 ° C (pH = 12-13). During this operation, impurities derived from trichloroacetamide were removed. Acidification of alkaline water fractions followed by extraction with methylene chloride confirmed that a very small amount of epoxymexrenone is lost during this operation.

The methylene chloride phase was washed once with 0.1 N hydrochloric acid (61 ml) for 15 minutes at a stirring speed of 250 rpm and at a temperature in the range of 15-25 ° C. Then these layers were allowed to separate and the lower methylene chloride layer was removed and washed again with 10% of the mass. aqueous sodium chloride (61 ml) for 15 minutes at a stirring speed of 250 rpm and at a temperature in the range of 15-25 ° C. Again, the layers were allowed to separate and the organic layer was removed. The organic layer was filtered through a Solkafloc layer, and then evaporated to dryness under reduced pressure. Drying was carried out in a water bath at a temperature of 65 ° C. The result was a whitish solid (17.95 g), which was subjected to HPLC analysis. The analysis showed that the yield of epoxymexrenone was 66.05%. The adjusted molar yield for this reaction was 93.1%.

The product was dissolved in hot methyl ethyl ketone (189 ml) and the resulting solution was distilled at atmospheric pressure until 95 ml of the ketone solvent was removed. The temperature was lowered to 50 ° C. as the product crystallized. Stirring was continued for 1 h at 50 ° C. Then the temperature was lowered to 20-25 ° C and stirring was continued for another 2 hours. The solid was filtered, washed with MEK (24 ml) and the solid was dried to obtain a constant weight of 9.98 g, an HPLC analysis of which indicated 93.63% epoxymexrenone in it. This product was again dissolved in hot MEK (106 ml) and the hot solution was filtered through a 10 micron line filter under pressure. Then, another 18 ml of MEK was applied as a wash and the filtered MEK solution was distilled at atmospheric pressure until 53 ml of solvent was removed. The temperature was lowered to 50 ° C. as the product crystallized; and stirring was continued at 50 ° C. for 1 hour. Then the temperature was lowered to 20-25 ° C and stirring continued at this temperature for another 2 hours. The solid was filtered and washed with MEK (18 ml). Then, the solid product was dried to obtain a constant mass of 8.32 g, which contained 99.6% epoxymexrenone, as indicated by quantitative HPLC analysis. The final loss after drying was less than 1.0%. The total yield of epoxymexrenone obtained by carrying out the reaction and treatment described in this Example was 65.8%. This overall yield was: reaction yield 93%, 78.9% after the first crystallization, and 89.5% after recrystallization.

Example 47B Preparation of 7-methylhydro-11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

Δ ^11,12- Olefin ester is a by-product of the elimination of 11-mesylate. From the reaction mixture obtained by the method described in Example 37A, a pure sample was isolated by repeated liquid chromatography. Thus, 73 g of the residue (obtained as described in Example 37A) was chromatographed on 2.41 kg of Merck silica gel (40-63 μm) according to an elution scheme using a gradient of ethyl acetate, toluene (20:80, 30:70, 40:60, 60:40 v / v). The Δ ^11,12 -olefin enriched portions were combined from the selected 30: 70 fractions. The appropriate fractions were selected based on TLC on EMF plates using ethyl acetate / toluene (60:40 v / v) and visualization with sulfuric acid SWUV. 7.9 g of crude Δ ^11,12 α-olefin (80% by HPLC area) obtained after solvent removal was chromatographed on 531 g of Merck silica gel (40-63 μm) according to an elution scheme using an ethyl acetate / methylene chloride gradient (10:90 , 20:80, 35:65 v / v). Pure 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,11-diene-7α, 21-dicarboxylate, γ-lactone (3.72 g) was obtained from the selected 20: 80 fractions. The choice of fractions was carried out based on TLC evaluation, as described above.

MICC ^-1 1767 (lactone), 1727 (ester), 1668 and 1616 (3-keto-Δ ^4,5 ).

¹ H-NMR (CDCl ₃ ) ppm 1.05 (s, 3H), 1.15 (s, 3H), 3.66 (s, 3H), 5.58 (dd, 1H), 5.80 (s, 1H), 5.88 (dd , 1H).

¹³ C-NMR (CDCl ₃ ) ppm 17.41; 18.58; 21.73; 28.61; 32.28; 33.63; 34.91; 35.64; 35.90; 38.79; 42.07; 44.12; 48.99; 49.18; 51.52; 93.81; 126.43; 126.69; 133.76; 166.24; 172.91; 176.64; 198.56.

A solution of 1.6 g (3.9 mmol) of 7-methylhydro-17-hydroxy-3-oxo-17α-pregna-4,11-diene-7α, 21-dicarboxylate, γ-lactone in 16 ml of methylene chloride was mixed with 2, 2 ml of trichloroacetonitrile (22.4 mmol) and 0.75 g of dipotassium phosphate (4.3 mmol). This mixture was stirred and combined with 6.7 ml of 30% hydrogen peroxide (66 mmol). Stirring was continued at 25 ° C. for 45 hours. After this time, 28 ml of methylene chloride and 39 ml of water were added. The organic part was isolated and washed sequentially: a) 74 ml of 3% sodium sulfate, b) 62 ml of 1n sodium hydroxide, c) 74 ml of 1N hydrochloric acid, and d) 31 ml of 10% saturated saline. The organic portion was again separated, dried with magnesium sulfate and evaporated to dryness in vacuo. 1.25 g of the residue was chromatographed over 138.2 g of Merck silica gel (40-63 μm) using a gradient system of methyl tert-butyl ether and toluene (40:60, 60:40, 75:25 v / v). The corresponding portions of the 60:40, 75: 25 fractions were combined on the basis of a TLC evaluation to obtain 0.66 g of pure 7-methyl-hydro-11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregn- 4-en-7α, 21-dicarboxylate, γ-lactone. EMF plates with an elution scheme with methyl tert-butyl ether and toluene 75:25 (v / v) and using sulfuric acid and SWUV for imaging were used in the TLC system.

¹ H-NMR (CDCl ₃ ) ppm 1.09 (s, 3H), 1.30 (s, 3H), 3.05 (AB ^11.12 for 2H), 3.67 (s, 3H), 5.80 (s, 1H).

¹³ C-NMR (CDCl ₃ ) ppm 14.2; 18.0; 21.2; 28.8; 31.9; 33.5; 34.6; 34.7; 35.1; 35.5; 37.4; 38.3; 41.8; 46.0; 47.2; 50.4; 51.7; 56.7; 94.0; 126.7; 165.2; 172.5; 176.7; 198.1

Calculated: C 69.54 and H 7.30; Found C, 69.29; and H, 7.17.

Example 47C Isolation of 7-methylhydro-4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone

The crude epoxymexrenone (157 g) obtained from 200 g of the ester by the method described in Example 26 was subjected to chromatography on 4.5 kg of Merck silica gel (40-63 μm). 88.1 g portion was isolated using an elution scheme with acetonitrile and toluene 10:90 (v / v). The isolated solid was dissolved in 880 ml of hot methyl ethyl ketone and filtered through a Solka Floc pad. An additional 88 ml of methyl ethyl ketone was then applied as a wash. The filtrate was concentrated by removing 643 ml of solvent, and the mixture was cooled to room temperature. The solids were filtered and washed with methyl ethyl ketone. After drying, 60.2 g of epoxymexrenone was obtained, which was 96.8% according to an HPLC analysis. The filtrate was concentrated to dryness under reduced pressure. 9.3 g of the residue were recrystallized from 99 ml of methyl ethyl ketone to obtain 2.4 g of a dry solid. A 400 mg portion of this solid was subjected to reverse phase preparative HPLC on a YMC ODS AQ column. Pure 7-methyl-hydro-4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone (103 mg) were isolated using an acetonitrile elution scheme (24 %), methanol (4%) and water (72%).

¹ H-NMR (CDCl ₃ ) ppm 0.98 (s, 3H), 1.32 (s, 3H), 2.89 (m, 1H), 3.07 (s, d, 2H), 3.73 (s, 3H).

MS, M + 430, calculated for C ₂₄ H ₃₀ O ₇ (430.50).

Example 47D Isolation of 7-methylhydro-17-hydroxy-3,12-dioxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone

The methyl ethyl ketone mother liquor obtained by the method described in Example 26 was evaporated to dryness under reduced pressure. 4.4 g of a portion of the residue was subjected to chromatography on 58.4 g of BTR Zorbax LP (40 μm). Elution with a gradient of methyl ethyl ketone and methylene chloride (25: 75-50: 50 v / v) gave 1.38 g of the product. 1.3 g of a portion of this product was further purified by reverse phase preparative HPLC using acetonitrile (30%), methanol (5%) and water (65%) and a YMC ODS AQ column (10 μm) as the mobile phase. This product was obtained from the enriched fractions by extraction with methylene chloride. Methylene chloride was evaporated to dryness and 175 mg of the residue was again purified by reverse phase preparative HPLC using acetonitrile (24%), methanol (4%) and water (72%) and a YMC ODS AQ column as mobile phase. Extraction of the enriched fractions with methylene chloride gave 30.6 mg of pure 7-methylhydro-17-hydroxy-3,12-dioxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

¹ H-NMR (CDCl ₃ ) ppm 1.17 (s, 3H), 1.49 (s, 3H), 3.13 (m, 1H), 3.62 (s, 3H), 5.77 (s, 1H), 5.96 (s , 1H).

¹³ C-NMR (CDCl ₃ ) ppm 13.1; 21.0; 28.0; 29.4; 33.1; 33.4; 33.9; 35.5; 36.7; 40.3; 41.5; 43.0; 43.4; 52.0; 55.0; 91.0; 123.7; 126.7; 163.2; 167.9; 171.8; 176.8; 197.4; 201.0.

Example 47E Preparation of 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, dihydrate, dipotassium salt

A suspension was prepared containing 2.0 g (4.8 mmol) of epoxymexrenone prepared as described in Example 43, 10 ml of water, 3 ml of dioxane and 9.3 ml of 1.04N aqueous potassium hydroxide (9.7 mmol). The mixture was stirred for 3 hours at 25 ° C. A yellow homogeneous solution formed during the first two hours. The temperature was raised to 70 ° C and stirring was continued for another 3 hours. The solvent was removed by vacuum distillation and the residue was purified by reverse phase HPLC on 90 g of C18 silica gel using water as an eluent. The desired fractions were combined according to the evaluation performed by TLC on EMF plates using methylene chloride and methanol (7: 3) as eluent and SWUV for visualization. The combined fractions were concentrated to dryness in vacuo and the residue was purified by reverse phase HPLC carried out as described above. The desired fractions were concentrated to dryness under reduced pressure, and the residue was dissolved in ethanol. Ethyl acetate was added to determine the cloud point, and then heptane was added to complete the precipitation. 0.55 g of product 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, dihydrate, dipotassium salt was isolated as a yellow solid. Carbon analysis corresponded to the hydrated structure of C ₂₃ H ₂₈ O ₇ K ₂ ^. 1.75 H ₂ O: Calculated C, 52.50 by avg. c 55.85 for the hydrated form; found C, 52.49. After TLC on EMF plates using methylene chloride, methanol and water (6: 3: 0.5 v / v) as an eluent and visualization using SWUV, R _f = 0.29 was observed.

Example 47F Preparation of 9.11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid, disodium salt

About 5 mg (0.01 mmol) of epoxymexrenone obtained by the method described in Example 43 was suspended in about 200 μl of methanol in a 4 ml vessel and diluted with about 200 μl of 2.5 NaOH. The resulting mixture was yellow and homogeneous. Then the mixture was heated in an oil bath at 70 ° C. After 10 minutes, 1 μm, a sample taken from this mixture was analyzed by HPLC (Zorbax SB-C8, 150 × 4.6 mm, 2 ml / min, gradient = 35:65 (v / v) A: B, A = acetonitrile / methanol (1: 1), B = water / 0.1% trifluoroacetic acid, detection at 210 nm) and this analysis revealed two products with retention times of 4.86 and 2.93 minutes, which corresponded to hydroxy acid (open lactone) and 7-carboxylic acid with an open lactone, respectively. After 30 minutes, a second sample (0.05 ml) was taken and acidified with 0.05 ml of 3 N HCl, and then neutralized using about 0.5 ml of sodium bicarbonate. HPLC analysis, as above, revealed the expected stained rings with closed rings at retention times of 6.59 and 10.71 minutes. The ratio of 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone (10.71 min) to the corresponding 7-carboxylic acid was 7 : 89.

Selective hydrolysis of lactone can be carried out under mild conditions. A second 4 ml vessel was prepared as described above, but without heating. The mixture was sonicated for 5 minutes. 0.05 ml of the sample was diluted in 0.5 ml of a methanol / acetonitrile mixture, 1: 1 (v / s), and analyzed by HPLC without prior acidification. The resulting open-lactone carboxylic acid 7-ester had a retention time of 4.85 minutes, as observed above, and had no impurities in the form of 7-carboxylic acid.

Example 47G Isolation of 7-methylhydro-9α, 11β, 17-trihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

and 7-methylhydro-12α, 17-dihydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone

7-Methylhydro-9α, 11β, 17-trihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone and 7-methylhydro-12α, 17-dihydroxy-3-oxo-17α- pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone was isolated after preparative liquid chromatography of a 2-butanone mother liquor obtained by epoxidation of the ester as described in Example 26 (scheme using trichloroacetonitrile) . For this, the first crystallization was carried out using the indicated 2-butanone. However, for recrystallization, 2-butanone (10 volumes per 1 g) was used instead of acetone. In this way, 2.8 g of residue was obtained, which was purified by reverse phase preparative HPLC. As the stationary phase, Cromasil C8 (10 μm) was used, and the mobile phase consisted of milliQ water and acetonitrile in a ratio of 70:30 (v / v). Crystallization was observed in one of the enriched fractions. A solid (46.7 mg) was isolated and identified as 7-methylhydro-9α, 11β, 17-trihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone. The mother liquor was evaporated to dryness under reduced pressure and the residue (123 mg) was identified as 7-methylhydro-12α, 17-dihydroxy-3-oxo-17α-pregna-4.9 (11) -diene-7α, 21-dicarboxylate, γ- lactone. 7-methylhydro-9α, 11β, 17-trihydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone:

MS M + 432, calculated for C ₂₄ H ₃₂ O ₅ (432.51).

¹ H-NMR (CDCl ₃ ) ppm 1.23 (s, 3H), 1.54 (s, 3H), 3.00 (m, 1H), 3.14 (m, 1H), 3.74 (s, 3H), 5.14 (s , 1H, slowly exchangeable), 5.79 (s, 1H).

¹³ C-NMR (CDCl ₃ ) ppm 16.8; 22.7; 24.8; 29.0; 29.3; 32.1; 34.1; 34.7; 35.2; 35.7; 36.8; 40.7; 43.0; 45.0; 45.9; 52.9; 72.8; 77.4; 95.9; 127.4; 163.7; 176.7; 177.3; 199.4.

7-methylhydro-12α, 17-dihydroxy-3-oxo-17α-pregna-4.9 (11) -diene-7α, 21-dicarboxylate, γ-lactone:

MS M + 441, calculated for C ₂₄ H ₃₀ O ₅ (414.50).

¹ H-NMR (CDCl ₃ ) ppm 0.87 (s, 1H), 1.40 (s, 1H), 3.05 (m, 1H), 3.63 (s, 3H), 3.99 (m, 1H); 5.72 (s, 1H); 5.96 (m, 1H).

¹³ C-NMR (CDCl ₃ ) ppm 14.8; 24.0; 26.1; 29.7; 33.6; 33.8; 34.0; 36.3; 37.0; 37.4; 40.7; 40.9; 43.8; 48.1; 51.9; 69.1; 95.5; 122.7; 126.3; 145.9; 164.5; 173.2; 177.6; 198.2.

Example 47H Preparation of 7-methylhydro-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

and 7-methylhydro-6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

7-Methylhydro-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone was obtained by the method described in RMWeier & LMHofmann (J.Med Chem. 1977, 1304), which is incorporated herein by reference. 148 g (357 mmol) of 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone obtained by the method described in Example 43, combined with 311 ml of absolute ethanol and 155 ml (932 mmol) of triethyl orthoformate. The suspension was stirred at room temperature and 10.4 g (54.7 mmol) of toluenesulfonic acid (monohydrate) was added as a catalyst. Stirring was continued for 30 minutes and the reaction was quenched by adding 41.4 g (505 mmol) of powdered sodium acetate and 20.7 ml (256 mmol) of pyridine. Solids (70.2 g) were removed by filtration and the filtrate was concentrated to dryness in vacuo. The residue was hydrolyzed with 300 ml of ethyl acetate and 9.8 g of solids were removed by filtration.

The filtrate was concentrated to dryness and the residue was hydrolyzed with 100 ml of methanol containing 2 ml of pyridine. 29.7 g of solids were removed by filtration. Additional precipitation was observed in the filtrate. Consequently, the filtrate was re-filtered to remove another 21.9 g of solids. The resulting filtrate was concentrated to dryness and the residue was hydrolyzed with 50 ml of methanol containing 1 ml of pyridine. 33.8 g of solids were isolated by filtration. Qualitative HPLC analysis indicated that this last portion of solids was reasonably pure (90% by area of 7-methylhydro-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone) for use in a subsequent reaction step.

7-Methylhydro-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone:

¹ H-NMR (CDCl ₃ ) ppm: 1.02 (s, 3H), 1.27 (s, 3H), 1.30 (t, 3H), 3.12 (m, 1H), 3 28 (m, 1H), 3.66 (s, 3H), 3.78 (m, 2H), 5.20 (s, 1H), 5.29 (d, 1H).

8 g portion of enol ester (7-methylhydro-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone) (18 mmol) obtained in the previous stage, dissolved in 120 ml of 1,4-dioxane. This solution was combined with a mixture of 6.8 g of 53% m-chloroperoxybenzoic acid (20.9 mmol), 18.5 ml of 1.0 N sodium hydroxide (18.5 mmol) and 46 ml of dioxane / water (9: 1). The temperature was maintained at -3 ° C and the mixture was stirred for two hours. Then the temperature was raised to 25 ° C and stirring was continued for another 20 hours. The mixture was combined with 400 ml of cold water (10 ° C) and 23.5 ml of 1.0 N sodium hydroxide (23.5 mmol). The mixture was extracted four times with 100 ml portions of methylene chloride (each time). The combined methylene chloride portions were dried with magnesium sulfate and the solvent of the supernatant was removed by vacuum distillation. 13.9 g of the residue was triturated with 50 ml of ethyl ether to give 2.9 g of a white solid. 2.4 g of a solid were chromatographed on 100 g of Merck silica gel (60 microns). After an initial wash of 1 L of ethyl acetate / heptane (1: 1), the product was eluted with 7: 3 ethyl acetate / heptane. The enriched fractions were combined based on a TLC evaluation (plates with EMF; eluent: ethyl acetate / heptane (7: 3 v / v); visualization using SWUV). Thus, 0.85 g of the enriched substance was obtained and recrystallized from 10 ml of isopropanol to obtain 0.7 g of 7-methylhydro-6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene 7α, 21-dicarboxylate, γ-lactone. Fractions with a large amount of impurities were combined and 0.87 g of crude 7-methylhydro-6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone was obtained . This product was chromatographed on 67.8 g of Merck silica gel (40-63 μm). Another 0.69 g of product was isolated using toluene containing 0.5-2.5% methanol. 7-methylhydro-6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone:

Calculated: C 66.96 and H 7.02: Found: C 66.68 and H 7.16.

¹ H-NMR (CDCl ₃ ): ppm: 1.06 (s, 3H), 1.36 (dm, 1H), 1.63 (s, 3H), 2.92 (m, 1H), 3.02 (dd, 1H), 3.12 (d, 1H), 3.64 (s, 3H), 4.61 (d, 1H), 5.96 (s, 1H).

¹³ C-NMR (CDCl ₃ ) ppm: 16.17; 21.32; 21.79; 24.36; 27.99; 28.94; 30.86; 31.09; 32.75; 33.19; 34.92; 36.77; 39.16; 43.98; 47.74; 51.56; 51.66; 65.36; 72.23; 94.79; 165.10; 171.36; 176.41, 199.59.

Example 47 I. Preparation of 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone

To 2 g (4.8 mmol) of 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone obtained by the method described in Example 43, 3.3 ml (14.4 mmol) of 25% sodium methoxide in methanol were added. The resulting yellow suspension was heated to 50 ° C. The solid did not dissolve. 3.3 ml of methanol (anhydrous methanol Aldrich) was added to the mixture. Then the mixture was heated under reflux (65 ° C) and this mixture became homogeneous. After 30 minutes, a solid precipitate interfered with stirring.

). Двуокись кремния элюировали 100 мл метиленхлорида. Затем элюированный метиленхлорид концентрировали в вакууме и получали 1 г коричневой пены, которая кристаллизовалась после добавления этилацетата. Слой двуокиси кремния элюировали второй раз 100 мл 10% этилацетатом/метиленхлоридом и элюированный раствор концентрировали, в результате чего получали 650 мг коричневой пены.Then, about 25 ml of anhydrous methanol was added and the mixture was transferred to a 100 ml flask. This mixture was heated under reflux for 16 hours, during which time it became dark and homogeneous. The mixture was cooled to 25 ° C. and 70 ml of 3N HCl (exothermic reaction) was added. To cool the mixture, several grams of ice was added and the solution was extracted with two consecutive 25 ml portions of methylene chloride. The dark solution was dried with sodium sulfate and filtered through a 2.5 cm layer of silica gel (E. Merck, 70-230 mesh, pore size 60

) Silica was eluted with 100 ml of methylene chloride. Then, the eluted methylene chloride was concentrated in vacuo to give 1 g of a brown foam, which crystallized after addition of ethyl acetate. The silica layer was eluted a second time with 100 ml of 10% ethyl acetate / methylene chloride and the eluted solution was concentrated, whereby 650 mg of a brown foam was obtained.

Thin layer chromatography (E. Merck, 60 F-254, silica gel 0.25 mm, toluene / ethyl acetate (1: 1 v / v)) revealed the presence of 7-methylhydro-9,11α-epoxy-17-hydroxy-3- oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone and 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7β, 21- dicarboxylate, γ-lactone in both samples, although a very small amount of the 7α-carboxy epimer was present in the first sample. The first sample was triturated with hot ethyl acetate (77 ° C) and allowed to cool to 25 ° C. The mixture was then filtered to obtain 400 mg of a whitish solid, mp. 254-258 ° C. H, ¹³ C and ¹³ C-ART confirmed the established structure. A small amount of ethyl acetate remained in the sample, but the presence of the starting compound was not detected by HPLC (Zorbax SB-C8, 150 × 4.6 mm, 2 ml / min, isocrate mixture = 40: 60 (v / v) A: B, A = acetonitrile / methanol (1: 1), B = water / 0.1% trifluoroacetic acid, detection at 210 nm) (HPLC showed 98.6% of the area) and TLC (toluene-ethyl acetate, 1: 1, vol. ./about.).

FAB-MC confirmed a molecular weight of 414 with M + H at 415.2.

¹ H-NMR (400 MHz, deuterochloroform) δ 0.95 (s, 3H), 1.50 (s, 3H), 1.45 (m, 3H), 1.55-2.7 (m, 15H) 2.85 (t, J = 13, 1H); 3.25 (d, J = 6, 1H); 3.65 (s, 3H); 5.78 (s, 1H).

Example 47J. Preparation of 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylic acid γ-lactone

K 774 mg (1.82 mmol) of 7-methylhydro-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-en-7α, 21-dicarboxylate, γ-lactone obtained by the method described in Example 43, and suspended in 3 ml of acetonitrile, 3 ml (7.5 mmol, 2.0 equivalents) of 2.5 M sodium hydroxide was added. The mixture turned yellow, and after 10 minutes it became homogeneous.

To monitor the progress of the reaction, aliquots (0.1 ml) of the mixture were quenched in 0.01 ml of 3M sulfuric acid and extracted in a 4 ml glass jar with ethyl acetate (0.2 ml). Phase separation was carried out by removing the lower aqueous phase with a pipette. The organic phase was evaporated and the residue was analyzed by HPLC by the method described in Example 47H. After 50 minutes at 25 ° C, a slight change in the composition of the mixture occurred.

The mixture was heated under reflux (about 90 ° C) for 50 minutes. An HPLC analysis of the mixture indicated the presence of 6% of the area of the remaining starting material. The mixture was stirred for 65 hours at 25 ° C. Acidification, extraction and HPLC analysis of an aliquot, as described above, confirmed the absence of remaining starting material.

The mixture was strongly acidified by adding about 4 ml of 3M sulfuric acid and extracted with two parts (about 10 ml) of methylene chloride. The organic phases were combined and dried with sodium sulfate. After concentration on a rotary evaporator, 780 g of solid was obtained. This solid was recrystallized from dimethylformamide / methanol to give 503 mg (67%) of a tan crystalline solid. Upon rapid heating, the sample was melted with evolution of gas at a temperature close to 260 ° C. Upon slow heating to 285 ° C, the sample slowly darkened, but remained solid.

¹ H-NMR (dimethyl sulfoxide d-6, 400 MHz) δ 0.85 (s, 3H), 1.4 (s, 3H), 1.3-2.9 (m, 19H), 3.15 (m , 1H), 5.55 (s, 1H), 11.8 (br., 1H).

Example 47K.

Scheme 1: Stage 3D: Method I: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

A 0.2 M solution of the ester of formula IIA in methylene chloride was combined with 2 equivalents of potassium bisphosphate dissolved in an equal weight of water (50% w / w aqueous solution), 3 equivalents of chlorodifluoroacetamide and 22 equivalents of hydrogen peroxide (added as 30% aqueous solution). Then the mixture was stirred at 25 ° C for 23 hours. The reaction mixture was diluted with an amount of water equal to the loading of hydrogen peroxide, and methylene chloride was separated. The methylene chloride portion was washed once with a 3% sodium sulfite solution (with a volume equal to 1.75 times the loading of hydrogen peroxide). The methylene chloride portion was separated and dried with sodium sulfate. The solution was concentrated by distillation at atmospheric pressure until the temperature in the head reached 70 ° C. The residue was evaluated by HPLC, ¹ H- and ¹ C-NMR (CDCl ₃ ). It was determined that the yield of epoxymexrenone was 54.2%, by area of HPLC.

Example 47L.

Scheme 1: Stage 3D: Method J: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

The method described in Example 47K was repeated, but heptafluorobutyramide (CF ₃ CF ₂ CF ₂ CONH ₂ ) was used instead of chlorodifluoroacetamide. The yield of epoxymexrenone was 58.4% by area HPLC.

Example 48. Epoxidation of the ester of formula IIA using toluene

Scheme 1: Stage 3D: Method K: Synthesis of methylhydro-9,11α-epoxy-17α-hydroxy-3-oxopregn-4-en-7α, 21-dicarboxylate, γ-lactone.

The ester of formula IIA was converted to epoxymexrenone by the method essentially described in Example 46, except that toluene was used as a solvent. Substances such as ester (2.7 g), trichloroacetamide (2.5 g), dipotassium bisphosphate (1.7 g), hydrogen peroxide (17.0 g) and toluene (50 ml) were charged into the reactor. The reaction was left to raise the temperature to 28 ° C and this reaction was completed after 4 hours. The resulting three-phase mixture was cooled to 15 ° C, filtered, washed with water and dried in vacuo to yield 2.5 g of product.

Example 49

Scheme 4: Method A: Epoxidation of 9,11-dienone

The compound designated XVIIA (compound XVII, where both -A-A- and -B-B- represent -CH ₂ -CH ₂ -) (40.67 g), was dissolved in methylene chloride (250 ml) in a one-liter 3-necked flask and then cooled outside with a mixture of ice and salt. Then, potassium bisphosphate (22.5 g) and trichloroacetonitrile (83.5 g) were added and the mixture was cooled to 2 ° C, after which 30% hydrogen peroxide (200 g) was slowly added over one hour. The reaction mixture was stirred at 12 ° C for 8 hours and at room temperature for 14 hours. Then, a drop of the organic layer was taken and evaluated for the presence of any starting enone and it was found to be <0.5%. After that, water (400 ml) was added, stirred for 15 minutes and the layers were separated. The organic layer was washed successively with 200 ml of potassium iodide (10%), 200 ml of sodium thiosulfate (10%) and 100 ml of saturated sodium bicarbonate solution, each time separating the layers. The organic layer was dried with anhydrous magnesium sulfate and concentrated to obtain a crude epoxide (41 g). This product was crystallized from ethyl acetate: methylene chloride to give 14.9 g of pure product.

Example 50

Scheme 4: Method B: Epoxidation of Compound XVIIA Using m-Chloroperoxybenzoic Acid

Compound XVIIA (18.0 g) was dissolved in 250 ml of methylene chloride and cooled to 10 ° C. Then, solid m-chloroperbenzoic acid (purity 50-60%; 21.86 g) was added over 15 minutes with stirring. No temperature increase was observed. The reaction mixture was stirred for 3 hours and evaluated for the presence of dienone. This reaction mixture was successively treated with a solution of sodium sulfite (10%), a solution of sodium hydroxide (0.5 N), a solution of hydrochloric acid (5%) and, finally, 50 ml of saturated saline. After drying with anhydrous magnesium sulfate and evaporation, 17.64 g of epoxide was obtained and used directly in the next step. This product was estimated to contain a Bayer-Williger oxidation product, which was removed by trituration with ethyl acetate, followed by crystallization from methylene chloride. Based on a scale of 500 g, precipitated m-chloroperbenzoic acid was filtered, followed by standard processing.

Example 51

Scheme 4: Method C: Epoxidation of Compound XVIIA Using Trichloroacetamide

Compound XVIIA (2 g) was dissolved in 25 ml of methylene chloride. Then trichloroacetamide (2 g) and dipotassium phosphate (2 g) were added. While stirring at room temperature, 30% hydrogen peroxide (10 ml) was added and stirring was continued for 18 hours to obtain an epoxide (1.63 g). The Bayer-Williger product did not form.

Example 52

Potassium hydroxide (56.39 g; 1005.03 mmol; 3.00 equiv.) Was charged into a 2000 ml flask and suspended with dimethyl sulfoxide (750.0 ml) at room temperature. Then, a trienone corresponding to the formula XX (where R ³ represents H and each of -A-A- and -B-B- represents -CH ₂ -CH ₂ -) (100) was charged into a flask together with THF (956.0 ml) (100 , 00 g; 335.01 mmol; 1.00 equiv.). After that, trimethylsulfonium methyl sulfate (126.14 g; 670.02 mmol; 2.00 equiv.) Was charged into the flask and the resulting mixture was heated under reflux for 1 h at 80-85 ° С. The conversion to 17-spirooxymethylene was monitored by HPLC. About 1 L of THF was evaporated from the reaction mixture under vacuum, after which water (460 ml) was charged for 30 minutes and the reaction mixture was cooled to 15 ° C. The resulting mixture was filtered and the solid oxirane product was washed twice with 200 ml aliquots of water. As was seen, the product became highly crystalline and it was easy to filter it. Then this product was dried in vacuum at 40 ° C. 104.6 g of the enol form 3-methyl ether of the Δ-5,6,9,11-17-oxiransteroid product were isolated.

Example 53

Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) Was charged into a dry 500 ml reactor in a stream of nitrogen. Then, ethanol (270.9 ml) was charged into the reactor and sodium methoxide was suspended in ethanol. Diethyl malonate (103.90 g; 648.68 mmol; 2.00 equiv.) Was charged into this suspension, followed by the addition of an oxirane steroid prepared as described in Example 52 (104.60 g; 324.34 mmol; 1.00 equiv.), and the resulting mixture was heated under reflux, that is, at 80-85 ° C. Heating was continued for 4 hours, after which completion of the reaction was monitored by HPLC. Water (337.86 ml) was charged into the reaction mixture for 30 minutes and the mixture was cooled to 15 ° C. Stirring was continued for 30 minutes, and then the reaction suspension was filtered to obtain a filter cake containing fine amorphous powder. This filter cake was washed twice with water (200 ml each) and then dried at room temperature under vacuum. 133.8 g of 3-methyl-enol-ether-Δ-5,6,9,11-17-spirolactone-21-ethoxycarbonyl intermediate were isolated.

Example 54

3-Methyl-enolether-Δ-5,6,9,11-17-spirolactone-21-ethoxycarbonyl intermediate (formula XVIII, where R ³ is H and each of -A-A- and -B-B- is -CH ₂ -CH ₂ -; 133.80 g; 313.68 mmol; 1.00 eq., Obtained as described in Example 53) together with sodium chloride (27.50 g; 470.52 mmol; 1.50 eq. .), dimethylformamide (709 ml) and water (5 ml) were loaded with stirring into a 2000 ml reactor. The resulting mixture was heated under reflux at 138-142 ° C for 3 hours, after which the reaction mixture was analyzed by HPLC to complete the reaction. Then, water was added to the mixture over 30 minutes, and the water was cooled to 15 ° C. Stirring was continued for 30 minutes, after which the reaction suspension was filtered, whereby an amorphous solid reaction product was isolated as a filter cake. This filter cake was washed twice (200 ml with aliquots of water) and then dried. After drying, 91.6 g of the product, 3-methylene-ether-17-spirolactone were obtained (yield 82.3%; 96%, area analysis).

Example 55

The enol ester obtained as described in Example 54 (91.60 g; 258.36 mmol; 1.00 eq.), Ethanol (250 ml), acetic acid (250 ml) and water (250 ml) were loaded into a 2000 ml reactor and the resulting suspension was heated under reflux for 2 hours. Then water (600 ml) was charged for 30 minutes and during this time the reaction mixture was cooled to 15 ° C. After that, the reaction suspension was filtered and the filter residue was washed twice with water (200 ml aliquots). Then the filter cake was dried; and received 84.4 g of the product, 3-keto Δ-4,5,9,11-17-spirolactone (compound of formula XVII, where R ³ is H, a -A-A- and -B-B- are -CH ₂ -CH ₂ -; yield 95.9%).

Example 56

Compound XVIIA (1 kg; 2.81 mol) was charged with tetrachloromethane (3.2 L) into a 4-necked 22-liter flask. N-bromosuccinamide (538 g) was added to the mixture, followed by acetonitrile (3.2 L). The resulting mixture was heated under reflux and maintained at this temperature at 68 ° C for approximately 3 hours, resulting in a clear orange solution. 5 minutes after heating, the solution became dark. After 6 hours, the heat source was removed and samples were taken of the mixture. The solvent was evaporated in vacuo and ethyl acetate (6 L) was added to the residue at the bottom of the vessel. The resulting mixture was stirred, after which a 5% sodium bicarbonate solution (4 L) was added and the mixture was stirred for 15 minutes, after which it was left to separate phases. The aqueous layer was removed and saturated brine (4 L) was added to the mixture, after which the mixture was stirred for 15 minutes. Then the mixture was again left to separate the phases and the organic layer was evaporated in vacuo to give a viscous suspension. Then dimethylformamide (4 L) was added and evaporation was continued at a vessel temperature of 55 ° C. The residue at the bottom of the vessel was allowed to stand overnight, and then DABCO (330 g) and lithium bromide (243 g) were added. Then the mixture was heated to 70 ° C. After an hour and a half heating, liquid chromatography samples were taken, and after heating for 3.50 hours, additional DABCO (40 g) was added. After heating for 4.5 hours, water (4 L) was added and the resulting mixture was cooled to 15 ° C. This suspension was filtered and the filter cake was washed with water (3 L) and dried on the filter overnight. The wet cake (978 g) was charged again into a 22 L flask and dimethylformamide (7 L) was added. The mixture thus obtained was heated to 105 ° C, after which the precipitate was completely dissolved in the solution. The heat source was removed and the mixture in the flask was stirred and cooled. Ice water was poured into the jacket of the reactor and the mixture inside the reactor was cooled to 14 ° C and held for 2 hours. The resulting suspension was filtered and washed twice with 2.5 L aliquots of water. The filter cake was dried in vacuo overnight. 510 g of a light brown solid were obtained.

Example 57

Into a 2 liter 4-necked flask were charged: 9.11-epoxycanrenone prepared as described in Example 56 (100.00 g; 282.1 mmol; 1.00 equiv.), Dimethylformamide (650.0 ml), lithium chloride (30.00 g; 707.7 mmol; 2.51 equiv.) And acetone cyanohydrin (72.04 g; 77.3 ml; 846.4 mmol; 3.00 equiv.). The resulting suspension was subjected to mechanical stirring and treated with tetramethylguanidine (45.49 g; 49.6 ml; 395.0 mmol; 1.40 equiv.). The system was then filtered using a water-cooled refrigerator and a dry ice refrigerator (filled with dry ice in acetone) to prevent HCN leakage. An exhaust pipe from a refrigerator with dry ice was attached to a scrubber filled with a significant excess of chlorine bleach. This mixture was heated to 80 ° C.

After 18 hours, a dark reddish-brown solution was obtained, which was cooled to room temperature with stirring. During cooling, the solution was sparged with nitrogen to remove residual HCN using a chimney going to the bleach scrubber. After 2 hours, the solution was treated with acetic acid (72 g) and stirred for 30 minutes. Then, the crude mixture was poured with stirring into ice water (2 L). Thereafter, the stirred suspension was treated with 10% aqueous HCl (400 ml) and stirred for 1 hour. The mixture was then filtered to give a dark brick red solid (73 g). The filtrate was placed in a 4 liter separatory funnel and extracted with methylene chloride (3 × 800 ml); and the organic layers were combined and back extracted with water (2 × 2 L). The methylene chloride solution was concentrated in vacuo to give 61 g of a dark red oil.

After the aqueous wash fractions were left overnight, a significant amount of precipitate formed. This precipitate was collected by filtration and it was determined that it was a pure enamine product (14.8 g).

After drying, the original red solid (73 g) was analyzed by HPLC and it was determined that the main component was 9,11-epoxyienamine. In addition, HPLC showed that enamine was the main component of the red oil obtained by treatment with methylene chloride. The calculated molar yield of enamine was 46%.

Example 58

9,11-epoxyienamine (4,600 g; 0.011261 mol; 1.00 eq.), Prepared as described in Example 57, was introduced into a round bottom 1000 ml flask. To the mixture was added methanol (300 ml) and 0.5% of the mass. aqueous HCl (192 ml), after which this mixture was heated under reflux for 17 hours. Then methanol was removed in vacuo, which led to a decrease in the amount of substance in the distillation cube to 50 ml and the formation of a white precipitate. Water (100 ml) was added to the suspension, and then it was filtered to obtain a white solid precipitate, which was washed three times with water. The yield of solid 9.11-epoxydiketone product was 3.747 g (81.3%).

Example 59

The epoxydiketone obtained by the method described in Example 58 (200 mg; 0.49 mmol) was suspended in methanol (3 ml) and 1.8-diazabicyclo [5.4.0] undec-7-ene (DBU) was added to the mixture. . After refluxing for 24 hours, the mixture became homogeneous. Then it was concentrated to dryness at 30 ° C on a rotary evaporator and the residue was partitioned between methylene chloride and 3.0N HCl. After concentrating the organic phase, a yellow solid (193 mg) was obtained, which was determined to be 22% by weight. epoxymexrenone. The yield was 20%.

Example 60

To 100 mg of diketone (prepared in accordance with Example 58) suspended in 1.5 ml of methanol was added 10 microliters (0.18 eq.) Of a 25% (w / w) solution of sodium methoxide in methanol. The solution was heated under reflux. After 30 minutes, no diketone was detected, but a 5-cyano ester was present. To the mixture was added 46 microliters of a 25% (w / w) methanol solution of sodium in methanol. The mixture was heated under reflux for 23 hours, during which HPLC revealed that the main product was epoxymexrenone.

Example 61

To 2 g of diketone (prepared according to Example 58) suspended in 30 ml of dry methanol, 0.34 ml of triethylamine was added. The suspension was heated under reflux for 4.5 hours. The mixture was stirred at 25 ° C. for 16 hours. The resulting suspension was filtered to give 1.3 g of 5-cyano ester as a white solid.

To 6.6 g of diketone suspended in 80 ml of methanol, 2.8 ml of triethylamine was added. The mixture was refluxed for 4 hours and stirred at 25 rpm for 88 hours, during which time the product crystallized from solution. As a result of filtration, and then washing with methanol, 5.8 g of a cyano ester was obtained in the form of a white powder. This powder was crystallized from chloroform / methanol to give 3.1 g of a crystalline product that was homogeneous, as indicated by HPLC.

Based on the foregoing, we can say that some of the objectives of the present invention were achieved and other advantageous results were obtained.

It should be noted that although various changes may be made in the above compositions and methods without departing from the scope of the present invention, however, all the options described in this application and illustrated in the accompanying drawings are illustrative and should not be construed as a limitation of the invention. .

New connections

In addition, the present invention relates to additional polycyclic organic molecules that can be used as chromatographic markers in the preparation of steroid compounds having predominant biological activity, such as spironolactone or epoxymexrenone.

In short, it has been found that certain compounds containing substituted or unsubstituted steroid nuclei and a substituted or unsubstituted carbocyclic ring fused to the steroid nucleus at the 13.17 position can be used as internal or chromatographic markers in the preparation of steroids such as spironolactone and epoxymexrenone. In particular, the compound methyl-2,3,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3aβ, 11aβ-dimethyl-1,9-dioxo-1H-pentaleno [1, 6a-a] phenanthrene-6α-carboxylate:

can be used as a chromatographic marker. One of the new distinguishing features of this compound and related compounds of the present invention is a fused carbocyclic ring attached to the D ring of the steroid nucleus. Spironolactone and epoxymexrenone do not have this feature, but instead they contain a 20-spirolactone ring.

Mentioned in the present description, the steroid core of this compound corresponds to the following structure:

This structure has the standard numbering and ring designations adopted for steroid compounds. The steroid core may be saturated, unsaturated, substituted or unsubstituted. It preferably contains at least one to four unsaturated bonds. More preferably, when each of the rings A, C and D contains at least one unsaturated bond. The steroid nucleus may also be substituted, as more specifically discussed below. This core is substituted, preferably with at least a C7 ester group.

Mentioned in the present description, the carbocyclic ring fused to the steroid core corresponds to a four-, five- or six-carbon cyclic skeleton. It may be saturated, unsaturated, substituted or unsubstituted. It is preferably saturated or has one double bond and is substituted with a hydroxy or keto group. In addition, the carbocyclic ring preferably has an α orientation with respect to the steroid nucleus.

In a preferred embodiment of the invention, the carbocyclic ring contains a five-carbon cyclic skeleton and has an α-orientation, and said compound is selected from the group consisting of compounds corresponding to the following formulas:

;

;

Where:

-A-A- represents the group -CR ¹⁰² R ^102a -CR ¹⁰³ R ^103a - or -CR ¹⁰² = CR ¹⁰³ , where -CR ¹⁰² R ^102a - and -CR ¹⁰² = corresponds to carbon C2, and CR ¹⁰³ R ^103a - and = CR ¹⁰³ - correspond to carbon C3;

—DD— represents the group —CHR ¹⁰⁴ —CH— or —CR ¹⁰⁴ = C—;

-E-E- represents a group -CH ₂ -CR ¹¹⁰ - or -CH = C;

-A-E- represents the group -CR ¹⁰² R ^102a -CH ₂ - or -CR ¹⁰² = CH, where -CR ¹⁰² R ^102a - and -CR ¹⁰² = correspond to carbon C2, and -CH ₂ - and = CH- correspond to carbon C1;

-GG- represents the group -CR ¹⁰⁶ R ^106a -CHR ¹⁰⁷ - or -CR ¹⁰⁶ = CR ¹⁰⁷ -, where -CR ¹⁰⁶ R ^106a - and -CR ¹⁰⁶ = correspond to carbon C6, and -CHR ¹⁰⁷ - and = CR ¹⁰⁷ - correspond carbon C7;

—JJ— represents the group —CR ¹⁰⁸ —CR ¹⁰⁹ - or —C = C—, where —CR ¹⁰⁸ — corresponds to carbon C8, and —CR ¹⁰⁹ — corresponds to carbon C9;

-LL- represents a group -CR ¹¹¹ R ^111a -CH ₂ - or -CR ¹¹¹ -CH-, where -CR ¹¹¹ R ^111a - and -CR ¹¹¹ = correspond to carbon С11, and -CHR ² - and = СН- correspond to carbon С12 ;

-JL- represents the group -CR ¹⁰⁹ -CR ¹¹¹ R ^111a - or -C = CR ¹¹¹ -, where -CR ¹⁰⁹ - and -C = correspond to carbon C9, and CR ¹¹¹ R ^111a - and -CR ¹¹¹ - correspond to carbon C11;

—M — M— represents the group —CR ¹¹⁴ —CH ₂ - or —C = CH—, where —CR ¹¹⁴ — and —C = correspond to carbon C14, and —CH ₂ - and —C = H— correspond to carbon C15;

JM- represents the group —CR ¹⁰⁸ —CR ¹¹⁴ - or —C = C—, where —CR ¹⁰⁸ — corresponds to carbon C8, a —CR ¹¹⁴ — corresponds to carbon C14;

-QQ- represents the group -CR ¹²⁰ R ^120a -CR ¹¹⁹ R ^119a - or -CR ¹²⁰ = CR ¹¹⁹ -, where -CR ¹²⁰ R ^120a - and -CR ¹²⁰ = CR ¹¹⁹ corresponds to carbon C20, and CR ¹¹⁹ R ^119a - and = CR ¹¹⁹ - correspond to carbon C19;

-QT- represents the group -CR ¹¹⁹ R ^119a -CHR ¹¹⁸ - or -CR ¹¹⁹ = CR ¹¹⁸ -, where -CR ¹¹⁹ R ^119a - and -CR ¹¹⁹ = correspond to carbon C19, and -CHR ¹¹⁸ - and = CR ¹¹⁸ - correspond carbon C18;

R ¹⁰² represents hydrogen, alkyl, alkenyl or alkynyl;

R ^102a represents hydrogen; or R ^102a represents a bond between a carbon atom C2 and a carbon atom C3 in the case where -A-A represents a group -CR ¹⁰² = CR ¹⁰³ -, and -A-E- represents a group -CR ¹⁰² R ^102a -CH ₂ -, or a bond between a carbon atom C1 and a carbon atom C2 if -A-E- represents a group -CR ¹⁰² = CH-, and -A-A- represents a group -CR ¹⁰² R ^102a -CR ¹⁰³ R ^103a -;

R ¹⁰³ represents hydrogen, hydroxy, protected hydroxy, R ¹³⁰ O—, R ¹³⁰ C (O) O—, R ¹³⁰ OC (O) O— or R ¹⁰³ taken together with R ^103a forms oxo with the proviso that —A— represents —CR ¹⁰² R ^102a —CR ¹⁰³ R ^103a - when R ¹⁰³ together with R ^103a form oxo;

R ^103a represents hydrogen, or R ^103a together with R ¹⁰³ form oxo with the proviso that -A-A- represents -CR ¹⁰² R ^102a -CR ¹⁰³ R ^103a - when R ^103a together with R ¹⁰³ form oxo;

R ¹⁰⁴ represents hydrogen, alkyl, alkenyl or alkynyl;

R ¹⁰⁶ represents hydrogen, hydroxy or protected hydroxy, either together with R ^106a forms oxo, or together with R ¹⁰⁶ and the carbon atom to which they are attached form a cyclopropyl, cyclobutyl or cyclopentyl ring, provided that -GG- represents a group -CR ¹⁰⁶ R ^106a —CR ¹⁰⁷ R ^107a - if R ¹⁰⁶ together with R ^106a form oxo;

R ^106a represents hydrogen, hydroxy or protected hydroxy, either together with R ¹⁰⁶ forms oxo, or together with R ¹⁰⁶ and the carbon atom to which they are attached forms a cyclopropyl, cyclobutyl or cyclopentyl ring; or R ^106a together with R ^107a and the carbon atoms to which they are attached form a cyclopropyl, cyclobutyl or cyclopentyl ring;

R ¹⁰⁷ represents hydrogen; hydroxycarbonyl; lower alkyl, alkenyl, alkynyl, aryl, heteroaryl or aralkyl; haloalkyl; hydroxyalkyl; alkoxyalkyl; lower alkanoyl, alkenoyl, alkynoyl, aryloyl, heteroaryloyl or aralkanoyl; lower alkoxycarbonyl, alkenoxycarbonyl, alkynoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl or aralkoxycarbonyl; lower alkanoylthio, alkenoylthio, alkynoylthio, aryloylthio, heteroroylthio or aralkanoylthio; lower alkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio or aralkylthio; carbamyl; alkoxycarbonylamino; or cyano; or

R ¹⁰⁷ together with R ^106a and the carbon atoms to which they are attached form a cyclopropyl, cyclobutyl or cyclopentyl ring; or

R ¹⁰⁷ and R ¹¹⁴ taken together with the carbon atoms C7, C8 and C14 form a γ-lactone;

R ¹⁰⁸ represents hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl, R ¹⁴⁰ O—, R ¹⁴⁰ C (O) O—, R ¹⁴⁰ OC (O) O—; or represents a bond between a C8 carbon atom and a C9 carbon atom if —JJ— represents a —C═C— group, and —JM— represents a —CR ¹⁰⁸ —CR ¹¹⁴ - group; or represents a bond between a C8 carbon atom and a C14 carbon atom if —JM— represents a —C═C— group and —JJ— represents a —CR ¹⁰⁸ —CR ¹¹⁴ - group;

R ¹⁰⁹ represents hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl, R ¹⁵⁰ O—, R ¹⁵⁰ C (O) O—, R ¹⁵⁰ OC (O) O—; or represents a bond between a C9 carbon atom and a C11 carbon atom if —JL— represents a —C═CR ¹¹¹ - group and —JJ— represents a —CR ¹⁰⁸ —CR ¹⁰⁹ - group; or represents a bond between a C9 carbon atom and a C8 carbon atom if —JJ— represents a —C═C— group, a —JL— represents a —CR ¹⁰⁹ —CR ¹¹¹ R ^111a - group;

R ¹¹⁰ represents hydrogen or methyl;

R ¹¹¹ represents hydrogen, hydroxy or protected hydroxy, or R ¹¹¹ together with R ^111a form oxo with the proviso that —JL— represents a group —CR ¹⁰⁹ —CR ¹¹¹ R ^111a -, a —LL— represents a group —CR ¹¹¹ R ^111a - CH ₂ -, if R ¹¹¹ together with R ^111a forms oxo;

R ^111a represents hydrogen, or together with R ¹¹¹ forms oxo, provided that -JL- represents a group -CR ¹⁰⁹ -CR ¹¹¹ R ^111a -, and -LL- represents a group -CR ¹¹¹ R ^111a -CH ₂ - if R ¹¹¹ together with R ^111a forms oxo; either R ^111a represents a bond between a C11 carbon atom and a C9 carbon atom if -JL- represents a —C = CR ¹¹¹ group, a -LL— represents a —CR ¹¹¹ R ^111a —CH ₂ - group, or R ^111a represents a bond between carbon atom C11 and carbon atom C12 in the case where LL-represents a group CR ¹¹¹ = CH, a -JL- represents a group CR ¹⁰⁹ R ^109a -CR ¹¹¹ R ^111a -;

R ¹¹⁴ represents hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl, R ¹⁶⁰ O—, R ¹⁶⁰ C (O) O—, R ¹⁶⁰ OC (O) O—; or R ¹¹⁴ and R ¹⁰⁷ together with the carbon atoms C7, C8 and C14 form a γ-lactone; or R ¹¹⁴ represents a bond between a C14 carbon atom and a C8 carbon atom if —JM— represents a —C═C— group and —M — M— represents a —CR ¹¹⁴ —CH ₂ - group; or R ¹¹⁴ represents a bond between a C14 carbon atom and a C15 carbon atom if —M — M— represents a —C═CH— group and —JM— represents a —CR ¹⁰⁸ —CR ¹¹⁴ - group;

R ¹¹⁸ represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylthio, alkenylthio or cyano;

R ^118a represents hydrogen or a bond between the C18 carbon atom and the C19 carbon atom in the case where —QT— represents the group —CR ¹¹⁸ = CR ¹¹⁹ -, and QQ— represents the group CR ¹¹⁹ R ^119a —CR ¹²⁰ R ^120a -;

R ¹¹⁹ represents hydrogen, alkyl or alkenyl;

R ^119a represents hydrogen or a bond between a carbon atom C19 and a carbon atom C20 in the case where -QQ- represents a group -CR ¹²⁰ = CR ¹¹⁹ -, and -QT- represents a group CR ¹¹⁹ R ^119a -CR ¹¹⁸ R ^118a -; or a bond between a carbon atom C19 and a carbon atom C18 in the case where -QT- represents a group -CR ¹¹⁹ = CR ¹¹⁸ -, a -QQ- represents a group CR ¹¹⁹ R ^119a -CR ¹²⁰ R ^120a -;

R ¹²⁰ represents hydrogen, hydroxy, protected hydroxy, or together with R ^120a forms oxo; with the proviso that —QQ— represents a group CR ¹¹⁹ R ^119a —CR ¹²⁰ R ^120a -, if R ¹²⁰ together with R ^120a form oxo;

R ^120a represents hydrogen, or together with R ¹²⁰ forms oxo; with the proviso that —QQ— represents a group CR ¹¹⁹ R ^119a —CR ¹²⁰ R ^120a -, if R ^120a together with R ¹²⁰ form oxo; and

R ¹³⁰ , R ¹⁴⁰ , R ¹⁵⁰ and R ¹⁶⁰ independently represent alkyl, alkenyl, alkynyl, aryl or heteroaryl.

More preferably, this compound corresponds to a compound of formula C-3, wherein R ¹⁰⁷ is hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoylthio, lower alkylthio, carbamyl, or R ¹⁰⁷ taken together with R ^106a and carbon atoms, with which they are connected, form a cyclopropyl ring; R ¹⁰⁶ represents hydrogen, hydroxy, or taken together with R ^106a and the carbon atom to which they are attached form a cyclopropyl ring; R ^106a represents hydrogen, hydroxy, or together with R ¹⁰⁶ and the carbon atom to which they are attached form a cyclopropyl ring; or R ^106a taken together with R ¹⁰⁷ and the carbon atoms to which they are attached form a cyclopropyl ring; a R ¹²⁰ represents keto.

The following definitions are used to discuss the 13.17-fused ring compounds described herein.

The term “lower alkyl” means an alkyl radical having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl, pentyl and hexyl. This radical may be straight, branched or cyclic, as well as substituted (in particular, aryl), unsubstituted or heterosubstituted.

The term “lower alkanoyl” means a radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms, and bonded to the parent molecular moiety through a carbonyl group. Formyl and acetyl are particularly preferred.

The term “lower alkoxycarbonyl” means a radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms and bonded to an oxygen atom, said oxygen atom being attached to the parent molecular moiety through a carbonyl group. Particularly preferred are methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl and n-hexyloxycarbonyl.

The term “lower alkenyl” means an alkenyl radical having from 2 to 6 carbon atoms, such as ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, sec-butenyl and tert-butenyl, pentenyl and hexenyl. This radical may be straight or branched, as well as substituted, unsubstituted or heterosubstituted. The terms "lower alkenoyl" and "lower alkenoxycarbonyl" are as defined for "lower alkanoyl" and "lower alkoxycarbonyl", respectively, except that they do not come from direct alkyl, but from direct alkenyl. Preferably, the oxygen bound to the alkenyl radical of any alkenoxycarbonyl group is separated from any unsaturated carbon by at least one methylene group.

The term "lower alkynyl" means an alkynyl radical having from 2 to 6 carbon atoms, such as ethynyl, propynyl, isopropinyl, butynyl, isobutynyl, sec-butynyl and tert-butynyl, pentynyl and hexynyl. This radical may be straight or branched, as well as substituted, unsubstituted or heterosubstituted. The terms "lower alkanoyl" and "lower alkynoxycarbonyl" are as defined for "lower alkanoyl" and "lower alkoxycarbonyl", respectively, except that they do not come from direct alkyl, but from direct alkynyl. Preferably, the oxygen bound to the alkynyl radical of any alkynoxycarbonyl group is separated from any unsaturated carbon by at least one methylene group.

The "aryl" part preferably contains, either individually or with various substituents, from 5 to 15 atoms and is phenyl.

The term “lower alkylthio” means a radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms, and bonded to the parent molecular moiety through a sulfur atom. Methylthio is particularly preferred.

The term “lower alkanoylthio” means a radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms, and attached to a carbonyl group, said carbonyl group being attached to the parent molecular moiety through a sulfur atom. Acetylthio is particularly preferred.

The terms "lower alkenylthio" and "lower alkenoylthio" are as defined for "lower alkylthio" and "lower alkanoylthio", respectively, except that they do not come from direct alkyl, but from direct alkenyl. Preferably, the sulfur atom of any alkenylthio group is separated from any unsaturated carbon by at least one methylene group.

The terms "lower alkynylthio" and "lower alkynoylthio" are as defined for "lower alkylthio" and "lower alkanoylthio", respectively, except that they do not come from direct alkyl, but from direct alkynyl. Preferably, the sulfur atom of any alkynylthio group is separated from any unsaturated carbon by at least one methylene group.

The term “carbamyl” means a —NH ₂ radical attached to the parent molecular moiety through a carbonyl group. The carbamyl group may be mono-substituted or di-substituted, and the substituents may be alkyl, alkenyl, alkynyl and aryl radicals.

The groups defined above may be unsubstituted or additionally substituted. Such additional substituents may be alkyl, alkenyl, alkynyl, aryl, heteroaryl, carboxy, such as alkoxy, carboxyalkyl, acyl, acyloxy, halogen, such as chlorine or fluorine, haloalkoxy, nitro, amino, amido and keto. The groups defined above, as well as additional substituents may also contain oxygen, sulfur, phosphorus and / or nitrogen.

As used herein, “Me” means methyl; "Et" means ethyl; and "Ac" means acetyl.

In an even more preferred embodiment of the present invention, the compound is selected from the group consisting of compounds having the following formulas:

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

; и

; and

where R ¹⁰⁷ , R ¹⁰⁶ , R ^106a and R ^{120 are} defined above. Preferably, R ¹⁰⁷ represents hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoylthio, lower alkylthio, carbamyl, or together with R ^106a and the carbon atoms to which they are attached forms a cyclopropyl ring; R ¹⁰⁶ represents hydrogen, hydroxy, or together with R ^106a and the carbon atom to which they are attached forms a cyclopropyl ring; R ^106a represents hydrogen, hydroxy, either together with R ¹⁰⁶ and the carbon atom to which they are bonded form a cyclopropyl ring, or together with R ¹⁰⁷ and the carbon atoms to which they are bonded form a cyclopropyl ring; a R ¹²⁰ represents keto. Even more preferably, if this compound, in addition, corresponds to the compound of formula C-5.

In an even more preferred embodiment, the compound of formula C-3 is selected from the group consisting of the following compounds:

In a most preferred embodiment, the compound is methyl 2,2,3a, 4,6,7,9,10, -11,11a, 12,13-dodecahydro-3αβ, 11αβ-dimethyl-1,9-dioxo 1H-pentaleno [1,6a-a] phenanthrene-6α-carboxylate:

This compound of formula C-1 is a particularly preferred chromatographic marker in the preparation of epoxymexrenone.

The novel compounds discussed herein can also be prepared in the form of their salts.

Getting new connections

In general, the novel compounds described immediately above can be prepared by reacting a steroid having a 20-spiroxane ring and the steroid core described above with trihalogenated alkanoic acid. A preferred reagent for this reaction is an alkali metal salt and an alkanoic acid used. It is particularly preferred that the reaction reagent includes 3-fluoroacetic acid and an alkali metal salt of this acid, such as potassium trifluoroacetate. In addition, a desiccant, such as trifluoroacetic anhydride, is preferably used in this reaction to reduce the level of free water present in the acid.

Steroid compounds used as starting compounds preferably have the following formula:

;

;

where -A-A-, -DD-, -E-E-, -A-E-, -GG-, -JJ-, -LL-, -JL-, -M-M-, -JM-, - QQ-and -QT- are defined above. Such starting compounds can be prepared and / or isolated by methods analogous to the methods described in Scheme 1 for the previously described method for the synthesis of epoxymexrenone. Alternatively, the starting compounds are commercially available.

The initial concentration of the steroid compound of formula C-3 is preferably at least about 0.1% by weight of the total reaction mixture, more preferably from about 2% to about 20% by weight, and even more preferably from about 5% to about 15% by weight . Preferably, the trihaloalkanoic acid is present in excess. If trifluoroacetic anhydride is used, its amount should be at least about 3% by weight of the total reaction mixture, more preferably from about 5% to about 25% by weight, and most preferably from about 10% to about 15% by weight.

In addition, the reaction temperature should exceed room temperature (22 ° C). Preferably, the reaction temperature is from about 40 ° C to 100 ° C, more preferably from about 50 ° C to 80 ° C, even more preferably from about 60 ° C to 70 ° C, and most preferably from about 60 ° C to 65 ° C. In this reaction, when the reaction temperature is raised to about 70 ° C. or higher, a higher amount of a by-product of C14 lactone is produced. The reaction time should be at least about 30 minutes, more preferably from about 30 to about 6 hours, even more preferably from about 45 minutes to about 4 hours, and most preferably from about one hour to two hours. In a preferred embodiment, the reaction time is from about one hour to two hours, and the reaction temperature is maintained at about 60 ° C.

Scheme S-1 below illustrates a particularly preferred embodiment of this method:

Scheme S-1.

Condensed ring steroids having different substituents at different positions of the steroid can be prepared according to the reaction schemes below. Additional procedures and methods that are not specifically described in this application and which can be used to introduce various substituents at different positions of the steroid are known to every person skilled in the art. The starting compounds can be either a steroid with a fused ring or a steroid with a 20-spiroxane ring. For ease of description, when the starting compound is a fused ring steroid, the following reaction schemes use specific steroids or groups of steroids as illustrative starting compounds. However, it should be noted that in the same series of reactions using another steroid with condensed rings as the starting compound, other steroid derivatives with condensed rings or their analogs can be produced. Similarly, for ease of description, when the starting compound is a steroid with a 20-spiroxane ring, some specific steroids with a 20-spiroxane ring are used as starting compounds. It should be noted that in the same series of reactions using another steroid with a 20-spiroxane ring as the starting compound, other steroid derivatives with a 20-spiroxane ring or their analogs can be produced.

Steroids having a carboxylic acid substituent at C7 can be obtained by saponification of a steroid having an alkoxycarbonyl substituent at C7, such as a compound of formula C-1. The saponification reaction can be carried out by treating the starting steroid with an alkaline reagent such as potassium or sodium hydroxide in a suitable solvent such as methanol, ethanol, isopropanol or the like, at temperatures up to the boiling point of the solvent in the presence or absence of water. As illustrated in Scheme S-2, the saponification reaction of a compound of formula C-1 gives a carboxylic acid of formula C-101.

Steroids having C7 non-methanoate carbon ester substituents can be prepared using carboxylic acids, such as C-101, as starting compounds. Treatment of such carboxylic acids with an alkylating agent such as an alkyl halide in the presence of a base (such as sodium bicarbonate, sodium carbonate, potassium bicarbonate or triethylamine) in a solvent such as dimethylformamide gives the desired esters. Examples of suitable alkylating agents are ethyl iodide, ethyl bromide, isopropyl iodide, hexyl iodide, benzyl bromide, allyl iodide and the like.

For the synthesis of carbamyls, a suitable starting compound is also C-101 carboxylic acid. Treatment of the acid with chloroformate, such as isobutyl chloroformate or ethyl chloroformate, in the presence of a base results in a mixed anhydride. Treatment of the mixed anhydride with an amine (such as dimethylamine, methylamine or benzeneamine) leads to the formation of carbamyl, where R ₁ and R ₂ are substituents on various amines.

Scheme S-2.

Some modifications at position C7 are made using unsaturated ketones, such as compounds of formula C-105 (shown below in Scheme S-3) as starting compounds. Sulfides are synthesized by addition of suitable thiols under basic conditions. Examples of suitable thiols are methyl mercaptan, ethyl mercaptan and the like. Suitable bases are piperidine, triethylamine and the like.

Treatment of unsaturated ketones (such as a compound of formula C-105) with thioalkanoic acids, such as thioacetic acid, leads to the formation of C7-thioacyl compounds, such as acetylthio.

A fused C6, C7 cyclopropyl substituent can be introduced by treating unsaturated ketones (such as a compound of formula C-105) with dimethyl sulfoxonium methylide, which is formed by treating trimethyl sulfoxonium halide with a suitable base (such as sodium hydride) in a suitable solvent.

These various synthesis schemes are illustrated in Scheme S-3 shown below:

Scheme S-3.

Steroids containing a C6 spirocyclopropyl ring are synthesized by the methods described below in Scheme S-4. First, enones, such as a compound of formula C-110, are protected as C3 enol ether by treatment with an ortho-ester such as triethylorthoformate or trimethylorthoformate in the presence of an acid such as p-toluenesulfonic acid. The resulting enol ether is treated with a Vilsmeier reagent formed in situ by adding phosphorus oxychloride to dimethylformamide to form a formyl compound, such as a compound of formula C-112. The reduction of the formyl group is carried out using a hydride reducing agent, such as lithium tri-tert-butoxyaluminum hydride in a solvent, such as tetrahydrofuran. This results in an intermediate alcohol which, after treatment with an acid, eliminates water to form a 6-methylene compound, such as a compound of formula C-113. A suitable acid is hydrochloric acid in an aqueous medium. Treatment of the 6-methylene compound with diazomethane leads to the formation of an intermediate pyrazoline, which decomposes after heating to produce a product, such as a spirocyclopropyl compound of formula C-114. A protected enol ether (such as a compound of the formula C-111) is a universal intermediate and treatment with a hydride reducing agent such as sodium borohydride followed by acid hydrolysis results in hydroxy compounds such as the compounds of the formula C-115 and C-116. These various synthesis steps are illustrated below in Scheme S-4:

Scheme S-4.

Steroids having a hydroxy substituent at C6 and an ester substituent at C7 can be synthesized by the methods illustrated in Scheme S-5 below. An ester (such as a compound of formula C-1) is protected at carbonyl C3 by forming a 3,5-dienol ether (such as a compound of formula C-117) using an ortho ester such as triethyl orthoformate or trimethyl orthoformate in the presence of an acid. A suitable acid is p-toluenesulfonic acid. Treatment of the enol ether with an oxidizing agent, such as meta-chloroperoxybenzoic acid, leads to the formation of a hydroxy compound, such as a compound of formula C-118.

Scheme S-5

Scheme S-6 illustrates the introduction of a double bond into the C1-C2 position of the steroid. This can be achieved by treating the desired steroid (such as the compounds of formulas C-1, C-108 and C-114) with a suitable oxidizing agent, such as dichlorodicyanobenzoquinone, in a suitable solvent (such as dioxane) at temperatures up to the boiling point. In this way, unsaturated compounds C1-C2, such as compounds of the formulas C-127, C-128 and C-129, can be prepared.

Scheme S-6.

Scheme S-7 illustrates the introduction of a double bond into a fused ring. A steroid (such as a compound of formula C-114) is treated with an orthoester (such as triethylorthoformate or trimethylorthoformate) in the presence of an acid catalyst (such as p-toluenesulfonic acid) to give an enol ether where C3 carbonyl is protected. In the case of a compound of the formula C-114, since the C6 position is completely substituted, the enol ether formed is an enol C2-C3 ether (such as a compound of the formula C-131). Treatment of the enol ester with a strong base such as lithium diisopropylamide at low temperature (-78 ° C to -30 ° C), followed by treatment with a selenizing agent such as phenyl selenyl chloride, leads to the formation of a selenium derivative, such as a compound of formula C-132 . Oxidation of the selenium derivative with an oxidizing agent such as hydrogen peroxide, for example, at room temperature, in the presence of a base such as pyridine, in a solvent such as methylene chloride, eliminates the selenium group and introduces a double bond. Hydrolysis of an enol ether results in a ketone, such as a compound of formula C-134.

Scheme S-7.

Scheme S-8 illustrates the synthesis of double bond isomers of a compound of formula C-1. Treatment of the various spiroxane compounds shown in Scheme S-8 with potassium acetate, trifluoroacetic anhydride and trifluoroacetic acid under conditions analogous to the synthesis conditions for the compounds of formula C-1 leads to the formation of compounds of formulas C-121 and C-123.

Scheme S-8.

Scheme S-9 illustrates an alternative method for the synthesis of double bond isomers for this steroid family. Condensed C6-C7-cyclopropane is introduced into a pre-prepared enone (such as a compound of formula C-24, the synthesis of which is described above) already containing a fused ring, using the chemical method described in Scheme S-3 above for the synthesis of compounds such as compounds formulas C-108 and C-109.

Scheme S-9.

where X is halogen.

Condensed ring steroids having aromatic ring A can be obtained by treating steroids, such as steroids, described by P. Compain, et al., Tetrahedron, 52 (31), 10405-10416 (1996) (which is introduced in the present description by reference), trifluoroacetic acid, potassium acetate and trifluoroacetic anhydride, generally in a manner similar to that previously described for Scheme S-1.

It is expected that these new fused ring steroids containing aromatic ring A and a 3-hydroxy substituent undergo all chemical reactions that are typical of phenols. Scheme S-10 illustrates the synthesis of 3-phenol ether from such a fused ring steroid. In particular, it is contemplated that treatment of these phenolic compounds with a base and an alkyl halide or alkyl sulfonate results in the production of the corresponding phenol ester. See Feuer & Hooz, In The Chemisty of the Ether Linkage, Patai (Ed.), Interscience: New York, pp. 466-450, 460-468 (1967); and Olson, W.T., J. Am. Chem. Soc., 69, 2451 (1947), which are incorporated herein by reference.

Scheme S-10.

Similarly, Scheme S-11 illustrates the synthesis of a 3-phenol ester from a fused ring steroid having an aromatic ring A and a 3-hydroxy substituent. In particular, it is contemplated that treatment of these phenolic compounds with a carboxylic acid anhydride or a carboxylic acid halide leads to the production of the corresponding phenol ester. See March, J., Advanced Organic Chemistry, Wiley: New York, pp. 346-347 (1985), which is incorporated herein by reference.

Scheme S-11.

Scheme S-12 illustrates the synthesis of 3-phenol carbonate from a fused ring steroid having an aromatic ring A and a 3-hydroxy substituent. In particular, it is believed that treatment of these phenolic compounds with an alkyl haloformate leads to the production of the corresponding phenol carbonate. See March, J., Advanced Organic Chemistry, Wiley: New York, pp. 346-347 (1985), which is incorporated herein by reference.

Scheme S-12.

Scheme S-13 illustrates the synthesis of ortho-allyl-substituted phenyl derivatives from a fused steroid having an aromatic ring A and a 3-hydroxy substituent. In particular, it is expected that treatment of these phenolic compounds with a base and an allyl halide leads to the production of the corresponding allyl phenyl ether. This allylphenyl ether should give a mixture of ortho-allyl-substituted phenyl derivatives after thermal rearrangement. See monograph by Shine, H., J. Arctic Rearrangements; Reaction Mechanisms in Organic Chemistry, Monograph 6, American Elsevier; New York, pp. 89-120 (1967), which is incorporated herein by reference.

Scheme S-13.

Scheme S-14 illustrates the synthesis of ortho-dialkyl substituted phenyl derivatives from a fused steroid having an aromatic ring A and a 3-hydroxy substituent. In particular, it is expected that treatment of these compounds with alcohol and acid leads to the production of the corresponding ortho-dialkylated phenyl derivatives. See, for example, Calcott, W.S., J. Am. Chem. Soc., 61, 1010 (1939), which is incorporated herein by reference.

Scheme S-14.

Scheme S-15 illustrates the allylic oxidation of a fused ring steroid having an aromatic ring A and a 3-hydroxy substituent. In particular, these compounds can be oxidized in an allyl position by reaction with selenium dioxide and tert-butyl hydroperoxide to form the corresponding alcohol. The dehydration of this alcohol gives the corresponding olefin. See, for example, Schmuff, N.R., J. Org. Chem., 48, 1404 (1983), which is incorporated herein by reference.

Scheme S-15.

Scheme S-16 illustrates the protection of the 3-carbonyl group and the reduction of the 20-carbonyl group of the new fused ring steroids of the present invention. In particular, these compounds can be reacted with a trialkyl orthoformate and an acid to form a 3-enol ether. This 3-enol ether can then be reacted with sodium borohydride to reduce the C-20 carbonyl group to the corresponding C-20 alcohol. Treatment of C-20 alcohol with acid and water provides the release of 3-enol ether to form a 3-keto derivative.

Scheme S-16.

Scheme S-17 illustrates the hydrogenation of olefin bonds in the novel fused ring steroids of the present invention. In particular, it is assumed that hydrogenation proceeds in stages. First, the C-6, C-7 double bond is saturated, and then the C-8, C-14 double bond is saturated.

Scheme S-17.

Scheme S-18 illustrates the rearrangement of the fused 11α-hydroxy steroids with fused rings of the present invention. In particular, first the 11α-hydroxy group is protected with a suitable protecting group such as 2-methoxyethoxymethyl ether (MEM ether). The treatment of the protected 11α-hydroxy steroid with an alkali metal salt and trihaloalkanoic acid in the presence of acid anhydride is expected to result in rearrangement of the lactone moiety in these molecules, as illustrated below. Removal of the MEM ester with zinc bromide leads to the formation of rearranged alcohols shown below.

Scheme S-18.

Scheme S-19 illustrates the protection of the 3-carbonyl group and the alkylation of the 19-position of the new fused ring steroids of the present invention. In particular, this compound is first converted to 3-alkyleneol ether, as shown in Scheme S-16. Treatment of 3-alkyleneol ether with lithium diisopropylamide (LDA) followed by treatment with an alkyl halide leads to the formation of a 19-alkyl derivative. As a result of hydrolysis of the protective group of a 3-alkyleneol ether, a 3-keto derivative is obtained.

Scheme S-19.

Scheme S-20 illustrates the conversion of estrone methyl ether to the corresponding spirolactone. Treatment of the lactone with trihalogenated alkanoic acid, preferably in the presence of an alkali metal salt of the used alkanoic acid, under the reaction conditions described previously, leads to the rearrangement of the lactone into the corresponding steroid with fused rings. See, for example, Otsubo, K., Tetrahedron Letters, 27 (47), 5763 (1986).

Scheme S-20.

The new compounds described in this application can also be subjected to biological conversion methods similar to the methods described previously, to obtain other new condensed ring steroids, such as steroids having 9α-, 9β-, 11α- or 11β- hydroxy substituent, as well as other hydroxylated steroids with fused rings. If necessary, such hydroxylated steroids can then be oxidized by eliminating the hydroxy substituent to introduce an olefinic double bond, such as a Δ ^9,11 -olefin double bond.

Based on the above, we can say that some of the objectives of the present invention were achieved and other advantageous results were obtained.

It should be noted that, although various changes may be made to the above compositions and methods without departing from the scope of the present invention, the entire material described in this application is illustrative and should not be construed as a limitation of the invention.

The following non-limiting examples illustrate various aspects of the present invention.

Example X-1A. Preparation of methyl 2,3,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3aβ, 11aβ-dimethyl-1,9-dioxo-1H-pentaleno [1,6a-a ] phenanthrene-6α-carboxylate (Compounds C-1)

Potassium acetate (6.7 g, 7.1 mmol; Sigma-Aldrich 5128LG) was added to a cleaned dry reactor equipped with a mechanical stirrer, a refrigerator, a thermocouple and a heating jacket. Then trifluoroacetic acid (25.0 ml, 8.1 mol; Sigma-Aldrich 7125MG) and trifluoroacetic anhydride (4.5 ml, 31.0 mmol; Sigma-Aldrich 11828PN) were successively added to the reactor. Then the solution was kept for 30 minutes at a temperature of 25 to 30 ° C.

The preformed reagent, TFA / TFA anhydride, was added to 5.0 g (9.6 mmol) of 7-methylhydro-17-hydroxy-11α- (methylsulfonyl) oxy-3-oxo-17α-pregna-4-en-7α, 21-dicarboxylate, γ-lactone:

which was obtained by the method described in Example 36. The resulting mixture was heated for 60 minutes at 60 ° C and the degree of conversion was periodically evaluated using TLC and / or HPLC. Upon completion of the reaction (approximately 60 minutes), the mixture was transferred to a 1-necked flask and concentrated under reduced pressure at 50 ° C until a thick suspension formed.

The resulting suspension was diluted with 150 ml of ethyl acetate and 80 ml of a mixture of water / saturated saline. The mixture was then allowed to separate the phases and the aqueous layer was re-extracted with 80 ml of ethyl acetate. The strength of the saturated saline solution was 12% of the mass. The combined ethyl acetate solution was washed once with 12% of the mass. saturated saline (80 ml), and then once with 1N NaOH solution (80 ml) and, finally, 2% of the mass. saturated saline (80 ml). The mixture was allowed to separate the phases and the separated ethyl acetate layer was concentrated to dryness under reduced pressure at 45 ° C. using a water suction device, whereby about 3.8 g of crude solid were obtained. HPLC analysis of this crude product showed that this product contained about 40% (by area) of compound C-1.

Then this solid product was subjected to chromatographic purification. After this chromatographic purification, 210 mg of methyl-2,3,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3aβ, 11aβ-dimethyl-1,9-dioxo-1H-pentaleno were obtained [1,6a-a] phenanthrene-6α-carboxylate (Compounds C-1).

Mass spectrometry data showed that the molecular weight is 380, and using the data obtained with high resolution, the formula C ₁₄ H ₂₈ O _{4 was established} . The mass spectrum obtained by the EI method had a M + peak at m / z 380. The mass spectrum obtained by the APHI method had peaks at m / z 381 (MN) + and m / z 398 (MNH ₄ ) +. Carbon and hydrogen analyzes were consistent with the proposed molecular formula.

The IR spectrum has two peaks in the region of carbonyl absorption: 1722 cm ^-1 and 1667 cm ^-1 . A peak of 1722 cm ^{-1 is} attributed to two carbonyls, since the ¹³ C-NMR spectrum had signals at δ 217.7 due to saturated ketone and at δ 172.7 due to carbomethoxycarbonyl. The absence of a peak of 1773 cm ⁻¹ in the IR spectrum indicates a loss of the lactone portion.

¹³ C-ART and HETCOR-NMR data indicate the presence of the following types of carbons: 3 carbonyls (δ 217.7, 198.4, 172.2); 4 fully protected olefinic carbon (δ 166.3, 141.8, 139.3, 121.8); 2 methinolefin carbon (δ 124.9, 122.0); 3 quaternary aliphatic carbon (δ 61.1, 50.7, 39.7); 1 methine aliphatic carbon (δ 43.3); 8 methylene carbon (δ 46.0, 37.5, 34.1, 33.3, 32.9, 31.9, 23.7, 22.2) and 3 methylene carbon (δ 51.9, 23.6 , 23.1).

Example X-1B. Preparation of (7α, 13R, 17β) -3 ', 4', 5 ', 17-tetrahydro-14-hydroxy-17-methyl-3,5'-dioxo-γ-lactone, cyclopent [13,17] -18- norandrosta-4.9 (11) -diene-7-carboxylic acid (Compounds C-201)

Potassium acetate (8.9 g, 90 mmol), trifluoroacetic acid (150 ml, 1.480 g / ml) and trifluoroacetic anhydride (33 ml, 1.487 g /) were added to a round-bottom 250 ml reactor equipped with a mechanical stirrer, a refrigerator and a heating jacket. ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25 ° C to 30 ° C.

The preformed reagent, TFA / TFA anhydride, was added to 15 g (30.0 mmol) of 7-methylhydro-17-hydroxy-11α- (methylsulfonyl) oxy-3-oxo-17α-pregna-4-en-7α, 21- dicarboxylate, γ-lactone:

which was obtained by the method described in Example 36. The resulting mixture was heated for about 1-1.5 hours at about 60 ° C-70 ° C. This mixture was concentrated under reduced pressure at 50 ° C. until a thick suspension formed. The resulting suspension was diluted with 100 ml of ethyl acetate and washed 2 times with approximately 20% water / saturated saline (each time 80 ml), 1 time with 1N sodium hydroxide solution (80 ml), and then 1 time with a mixture of approximately 20% water / saturated saline ( 80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated, whereby about 18 g of crude material was obtained.

This product was purified twice by column chromatography to give about 3 g of pure (7α, 13R, 17β) -3 ', 4', 5 ', 17-tetrahydro-14-hydroxy-17-methyl-3,5'-dioxo γ-lactone, cyclopenta [13,17] -18-norandrosta-4,9 (11) -diene-7-carboxylic acid (Compounds C-201).

Example X-1C. Preparation of [13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] gon-1,3,5 (10) -trien-5' - [2'H] -one (Compounds C-202)

Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid (150 ml, 1.480 g / ml) and trifluoroacetic anhydride (26 ml, 1.487 g /) were added to a round-bottom 250 ml reactor equipped with a mechanical stirrer, a refrigerator and a heating jacket. ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25 ° C to 30 ° C.

The preformed reagent, TFA / TFA anhydride, was added to 15 g (43.7 mmol) of 17-hydroxy-3-oxo-17α-pregn-4-ene-21-carboxylic acid, γ-lactone (also known as aldone; GD Searle & Co.):

The resulting mixture was heated for about 1-1.5 hours at 60 ° C-70 ° C. This mixture was concentrated under reduced pressure at 50 ° C. until a thick suspension formed. The resulting suspension was dissolved in 100 ml of ethyl acetate and washed 2 times with approximately 20% water / saturated saline (each time 80 ml), 1 time with 1N sodium hydroxide solution (80 ml), and then 1 time with a mixture of approximately 20% water / saturated saline (80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated to dryness under reduced pressure at 50 ° C., whereby about 20 g of a crude wet product was obtained.

This product was purified twice by column chromatography to give about 125 g of pure [13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] rut-1,3,5 (10) -trien-5 '- [2'H] -one (Compound C-202).

Example X-1D. Preparation of [13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] gon-1,3,5 (10), 6-tetraen-5' - [2 ' H] -one (Compounds C-203)

Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid (150 ml, 1.480 g / ml) and trifluoroacetic anhydride (26 ml, 1.487 g /) were added to a round-bottom 250 ml reactor equipped with a mechanical stirrer, a refrigerator and a heating jacket. ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25 ° C to about 30 ° C.

The preformed reagent, TFA / TFA anhydride, was added to 15 g (45.9 mmol) of 17-hydroxy-3-oxo-17α-pregn-4,9 (11) -diene-21-carboxylic acid, γ-lactone (known also as Δ-9,11-aldone):

which was obtained from 3-methoxy-3,5,9 (11) -androstatrien-17-one (Upjohn). The resulting mixture was heated at about 60 ° C-70 ° C for about 1-1.5 hours. This mixture was concentrated under reduced pressure at 50 ° C. until a thick suspension formed. The resulting suspension was diluted with 100 ml of ethyl acetate and washed 2 times with a mixture of approximately 20% water / saturated saline (each time 80 ml), 1 time with 1N sodium hydroxide solution (80 ml), and then 1 time with approximately 20% water / saturated saline ( 80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated to dryness under reduced pressure at 50 ° C., whereby about 18 g of a crude wet product was obtained.

This product was purified twice by column chromatography to give about 340 g of pure [13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] rut-1,3,5 (10) 6-tetraen-5 '[2'H] -one (Compounds C-203).

Example X-1E. Preparation of [13S, 17β] -3 ', 4'-dihydro-17-methylcyclopenta- [13.17] -18-norandrosta-4,6,8 (14) -trien-3,5' [2'H] - dione (Compounds C-204)

Potassium acetate (8 g, 81.5 mmol), trifluoroacetic acid (150 ml, 1.480 g / ml) and trifluoroacetic anhydride (33 ml, 1.487 g /) were added to a round-bottomed 250 ml reactor equipped with a mechanical stirrer, a refrigerator and a heating jacket. ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25 ° C to about 30 ° C.

The preformed reagent, TFA / TFA anhydride, was added to 15 g (44.0 mmol) of 17-hydroxy-3-oxo-17α-pregn-4,6-diene-21-carboxylic acid, γ-lactone (also known as canrenone ; GDSearle & Co.):

The resulting mixture was heated for about 1-1.5 hours at about 60 ° C-70 ° C. This mixture was concentrated under reduced pressure at 50 ° C. until a thick suspension formed. The resulting suspension was dissolved in 100 ml of ethyl acetate and washed 2 times with a mixture of approximately 20% water / saturated saline (each time 80 ml), 1 time with 1N sodium hydroxide solution (80 ml), and then 1 time with a mixture of approximately 20% water / saturated saline solution (80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated to dryness under reduced pressure at 50 ° C., whereby about 18 g of a crude wet product was obtained.

This product was purified twice by column chromatography to give about 2.2 g of pure [13S, 17β] -3 ', 4'-dihydro-17-methylcyclopenta- [13,17] -18-norandrost-4,6,8 (14) -trien-3,5 '[2'H] -dione (Compound C-204).

Example X-2. Receiving:

To a stirred cold (0 ° C) solution of 11α-hydroxycanrenone (3.6 g, 10 mmol) and triethylamine (1.2 g, 12 mmol) in methylene chloride (20 ml) was added methanesulfonyl chloride (1.1 g, 10 mmol). The mixture was stirred while cooling for 3 hours and left to warm to room temperature. Stirring was continued until thin layer chromatography indicated completion of the reaction. The mixture was then diluted with ethyl acetate and extracted with water, aqueous 5% sodium bicarbonate and water, and then dried with sodium sulfate. The desiccant was filtered and the filtrate was concentrated in vacuo to give crude C-136 mesylate, which can be used in the next step:

The C-136 mesylate solution (4.3 g, 10 mmol) was reacted with trifluoroacetic acid (25 ml), trifluoroacetic anhydride (4.5 ml) and potassium acetate (6.7 g, 7.1 mmol) in the manner described for for the synthesis of Compound C-1 in Example X-1. The crude product was isolated by a method similar to that described for Compound C-1 in Example X-1 and purified by silica gel chromatography using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents. Then, the resulting product was purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane to give C-105 tetraene.

Example X-3. Getting connection:

Mesylate S-138:

were synthesized and isolated (by the method described in Example X-2 for the synthesis of C-136 mesylate) using 11α, 17-dihydroxy-3-oxo-17-oxo-pregn-4-en-21-carboxylic acid, γ-lactone ( 3.6 g, 10 mmol), triethylamine (1.2 g, 12 mol) and methanesulfonyl chloride (1.1 g, 10 mmol) in methylene chloride (20 ml). The C-138 mesylate thus isolated can be used in the next step.

The C-138 mesylate solution (4.4 g, 10 mmol) was reacted with trifluoroacetic acid (25 ml), trifluoroacetic anhydride (4.5 ml) and potassium acetate (6.7 g, 7.1 mmol) in the manner described for for the synthesis of Compound C-1 in Example X-1. The crude C-110 product was isolated by a method similar to that described for Compound C-1 in Example X-1, and purified by silica gel chromatography using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents. Then, the resulting product was purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane.

Example X-4. Preparation of 3 ′, 4 ′, 5 ′, 17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta [13R, 17] -18-norandrosta-4,8,14-triene-7α-carboxylic acid (Compounds C- 101)

A solution of Compound C-1 (3.8 g, 10 mmol) and a 1N aqueous solution of sodium hydroxide (35 ml) in ethanol (60 ml) was heated under reflux for 8 hours. The reaction mixture was cooled to room temperature, concentrated on a rotary evaporator in vacuo, and the residual aqueous layer was extracted three times with ethyl acetate. The aqueous layer was then acidified with 1N hydrochloric acid and extracted three times with ethyl acetate. The combined organic layers were washed with water and dried with sodium sulfate. The desiccant was filtered and the filtrate was concentrated on a rotary evaporator. The residual crude C-101 carboxylic acid was crystallized by treatment with ethyl acetate and recrystallized from ethyl acetate and hexane or methanol or ethanol and water.

Example X-5. Preparation of 1-methylethyl-3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norandrosta-4,8,14-triene-7α-carboxylate ( Compounds C-102)

A mixture of sodium bicarbonate (3.5 g) and a solution of C-101 carboxylic acid (3.7 g, 10 mmol) and isopropyl iodide (3 ml) in dimethylformamide (35 ml) was stirred overnight at room temperature. The reaction mixture was poured into water and the aqueous solution was extracted three times with ethyl acetate. The combined organic layers were washed with water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo. The resulting crude C-102 isopropyl ester was crystallized by treatment with ethyl acetate or alcohol and purified by silica gel chromatography, and then recrystallized from ethyl acetate and hexane, or alcohol, or alcohol, and water.

Example X-6. Preparation of ethyl 3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norandrosta-4,8,14-triene-7α-carboxylate

A mixture of sodium bicarbonate (3.5 g) and a solution of C-101 carboxylic acid (3.7 g, 10 mmol) and ethyl iodide (3 ml) in dimethylformamide (35 ml) were stirred overnight at room temperature. The reaction mixture was poured into water and the aqueous solution was extracted three times with ethyl acetate. The combined organic layers were washed with water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo. The residual crude ethyl ester C-103 was crystallized by treatment with ethyl acetate or alcohol and purified by silica gel chromatography, and then recrystallized from ethyl acetate and hexane, or alcohol, or alcohol, and water.

Example X-7. Preparation of Hexyl-3 ′, 4 ′, 5 ′, 17-tetrahydro-17β-methyl-3,5′-dioxocyclopenta [13R, 17] -18-norandrosta-4,8,14-triene-7α-carboxylate (Compounds C -104)

A mixture of sodium bicarbonate (3.5 g) and a solution of C-101 carboxylic acid (3.7 g, 10 mmol) and n-hexyl iodide (3 ml) in dimethylformamide (35 ml) was stirred overnight at room temperature. The reaction mixture was poured into water and the aqueous solution was extracted three times with ethyl acetate. The combined organic layers were washed with water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo. The residual crude C-104 n-hexyl ester was crystallized by treatment with ethyl acetate or alcohol and purified by silica gel chromatography and then recrystallized from ethyl acetate and hexane, either from alcohol or from alcohol and water.

Example X-8. Preparation of 3 ', 4'-dihydro-17-methyl-7α (methylthio) -cyclopenta [13,17] -18-norandrost-4,8,14-triene-3,5' (2'H) -dione (Compounds S-106)

A solution of tetraene C-105 (3.2 g, 10 mmol) in methanol (40 ml) and piperidine (4 ml) was cooled to 5 ° C. Then, gaseous methyl mercaptan was passed until an increase of 7 g was observed. The pressure vessel was sealed and kept at room temperature for 20 hours. Then the solution was poured into ice water and the precipitate was filtered, washed with water and dried in air. The methylthioproduct C-106 was purified by recrystallization from methanol or ethyl acetate and hexane. See, for example, the procedure described by A. Karim & E. A. Brown, Steroids, 20, 41 (1972), which is incorporated herein by reference.

Example X-9. Preparation of 7α- (acetylthio) -3,4′-dihydro-17-methylcyclopenta [13,17] -18-norandrosta-4,8,14-triene-3,5 '(2'H) -dione (Compounds C- 107)

A solution of tetraene C-105 (3.2 g, 10 mmol) in thioacetic acid (10 ml) was heated for 1 hour at 85-95 ° C. Then the excess thioacetic acid was removed in vacuo and the resulting crude C-107 7α-thioacetate was purified by recrystallization from a suitable solvent such as methanol or ethyl acetate or ethyl acetate and hexane. See, for example, the procedure described in US Pat. No. 3,013,012, J. A. Colella & R. C. Twweit, Dec 12, 1961, which is incorporated herein by reference.

Example X-10. Preparation of 1,2,4bR (4bR *), 5,5aS *, 7,7aR *, 8,9,11, -12bS * -decahydro-7a, 12b-dimethyl-10aR * -cyclopropal [1] pentaleno [1, 6a-a] phenanthrene-3,10-dione

and

1,2,4bS (4bR *), 5,5аS *, 8,9,11,12,12bR * -dedecahydro-7a, 12b-dimethyl-10aS * -cyclopropal [1] pentaleno [1,6a-a] phenanthrene -3,10-dione (Compounds C-109):

To a solution of trimethyl sulfoxonium iodide (1 g, 4.6 mmol) in dry dimethyl sulfoxide (20 ml) was added sodium hydride (220 mg of a 50% dispersion in mineral oil, 4.6 mmol). The mixture was stirred at room temperature under a nitrogen atmosphere until hydrogen evolution ceased. Then, a solution of C-105 tetraene (1.12 g, 3.5 mmol) in dimethyl sulfoxide (4 ml) was added and stirring was continued for 4 hours under nitrogen. The reaction mixture was diluted with water and the resulting precipitate was filtered and dried in air. The resulting product was a mixture of 6β, 7β- (Compound C-108) and 6α, 7α- (Compound C-109) isomers. These isomers were isolated by silica gel chromatography, and then the individual isomers were purified by recrystallization from solvents such as ethyl acetate and hexane, alcohol or alcohol, and water.

Example X-11. Obtaining a compound of the formula:

To a suspension of enone C-110 (3.2 g, 10 mmol) in ethyl orthoformate (10 ml) and anhydrous ethanol (10 ml) was added p-toluenesulfonic acid monohydrate (0.05 g). The reaction mixture was stirred for 30 minutes at room temperature and quenched by adding a few drops of pyridine. After stirring for another 5 minutes at 0 ° C, the resulting precipitate was filtered, washed with a small amount of methanol and recrystallized from alcohol or ethyl acetate and hexane containing trace amounts of pyridine, resulting in a pure enol ether C-111. Alternatively, after the addition of pyridine, this reaction mixture can be treated by removing all solvents in vacuo and crystallizing the residue by adding solvents such as ether, ethyl acetate or hexane. Crude C-111 was recrystallized as described above. See, for example, the procedure described in R. M. Weier & L. M. Hofmann, J. Med. Chem., 20, 1304-1303 (1977), which is incorporated herein by reference.

Example X-12. Getting connection:

The procedure described in R. M. Weier & L. M. Hofmann, J. Med. Chem., 20, 1304-1308 (1977), which is incorporated herein by reference.

Vilsmeier reagent was obtained by adding phosphorus oxychloride (4.59 g, 30 mmol) to dimethylformamide (30 ml) at 0 ° C. After 5 minutes, a solution of enol ether C-111 (3.5 g, 10 mmol) in dimethylformamide (5 ml) was added and the reaction mixture was stirred for 2 hours at 0 ° C and overnight at room temperature. The reaction mixture was poured into a solution of aqueous sodium acetate and stirred for 2 hours. The precipitate was filtered and dried, resulting in a crude aldehyde C-112. Purification was carried out by recrystallization from solvents such as alcohol, alcohol and water, or ethyl acetate and hexane. Alternatively, the crude C-112 aldehyde was isolated from an aqueous solution of sodium acetate by extraction with a solvent such as ethyl acetate. After drying with sodium sulfate and removing the solvent, the residue was purified by recrystallization or chromatography on silica gel, followed by recrystallization.

Example X-13. Getting connection:

To a stirred, cooled solution of lithium tri-tert-butoxy-aluminum hydride (3.05 g, 12 mmol) in tetrahydrofuran was added C-112 aldehyde (3.78 g, 10 mmol). The reaction mixture was stirred at room temperature for 5 hours and quenched by adding water and acetic acid, while making sure that the mixture remained slightly basic. This mixture was concentrated in vacuo and the resulting solid was suspended in ethyl acetate. The solid was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in a minimum volume of a solution of acetone and water (3: 1) and aqueous acetone was added to acidify (pH 1.5-2.0). The acidic reaction mixture was stirred for 1 hour at room temperature and concentrated in vacuo. The resulting crude C-113 dienone was purified by recrystallization from solvents such as alcohol, alcohol and water, or ethyl acetate and hexane. Alternatively, the crude C-113 dienone was purified by silica gel chromatography and then recrystallized. See, for example, the procedure described in P.M. Weier & L.M. Hofmann, J.Med.Chem., 20, 1304-1308 (1977), which is incorporated herein by reference.

Example X-14. Preparation of 2 ', 3', 3'aα, 4 ', 6', 10 ', 11', 11'aα, 12 ', 13'-decahydro-3'aR, 3'a, 11'a-dimethyl-13 'aR * -spiro- [cyclopropane-1,7' (9'H) - [1H] pentaleno [1,6a-a] -phenanthrene-1'9'-dione:

To a solution of C-113 dienone (1.17 g, 0.05 mmol) in tetrahydrofuran (30 ml) was added a solution of diazomethane (0.2 g, 0.07 mmol) in ether (7 ml). The resulting reaction solution was stored at room temperature for several days. Then acetic acid was added to decompose the residual diazomethane and the reaction mixture was concentrated in vacuo. The residue, which was an intermediate pyrazoline, was crystallized from a solvent such as acetone, hexane, ethyl acetate or ethanol, and then recrystallized. This compound was converted to spirocyclopropane C-114. The C-114 solid thus obtained was heated at 190 ° C. in vacuo, and the obtained solid was recrystallized from alcohol, alcohol and water, acetone and water, or ethyl acetate and hexane. Alternatively, a solution of pyrazoline in acetone was treated with boron trifluoride etherate for about 1 hour at room temperature. Then water was added and the resulting precipitate was filtered and dried in air. Purification was carried out by recrystallization. See, for example, the procedure described in US Pat. No. 3,499,891 issued March 10 to F. B. Colton & R. T. Nicholson, which is incorporated herein by reference.

Example X-15. Getting connection:

and

Sodium borohydride (38 mg) was added to a solution of enol C-111 ether (3.2 g, 10 mmol) in methanol (20 ml). The reaction mixture was stirred for 3 hours at room temperature and treated with 1N hydrochloric acid for 30 minutes. Then the reaction mixture was diluted with water and the precipitate was filtered, washed with water and dried. The product, consisting of a mixture of two epimeric alcohols (Compounds C-115 and Compounds C-116), was chromatographed on silica gel to separate them. Each of the purified C-115 and C-116 alcohols was crystallized from solvents such as alcohol, alcohol and water, ethyl acetate and hexane, and acetone and hexane. Alternatively, the diluted reaction mixture was extracted with ethyl acetate, the combined organic layers were dried with sodium sulfate, and the crude product thus isolated was chromatographed as described above to give separate C-115 and C-116 alcohols.

Example X-16. Getting connection:

Compound C-1 (3.8 g, 10 mmol) was converted to C-117 enol ether using ethyl orthoformate (3.2 g, 10 mmol), ethanol (10 ml) and p-toluenesulfonic acid monohydrate (0.05 g ) in accordance with the procedure described for the synthesis of Compound C-111 in Example X-11.

Example X-17. Preparation of methyl-3 ′, 4 ′, 5 ′, 17-tetrahydro-6α-hydroxy-17β-methyl-3,5′-dioxocyclopenta- [13R, 17] -18-norandrost-4,8,14-triene-7α β-carboxylate (Compounds C-118):

A solution of 57% m-chloroperoxybenzoic acid (3.64 g) in 10% aqueous dioxane (20 ml) was half neutralized with a 1N sodium hydroxide solution. This solution was cooled to 0 ° C and a cold (0 ° C) solution of enol ether C-117 (4.1 g, 10 mmol) in 10% aqueous dioxane (20 ml) was added portionwise with stirring. The reaction mixture was stirred overnight at room temperature, poured into ice water and extracted with methylene chloride or ethyl acetate. The combined organic layers were dried with sodium sulfate. After evaporation of the solvent, a crude C-118 hydroxy ester was obtained, which was purified by silica gel chromatography. See, for example, the procedure described in R. M. Weier & L. M. Hofmann, J. Med. Chem., 20, 1304-1308 (1977), which is incorporated herein by reference.

Example X-18. Getting connection:

Lithium dimethyl cuprate was obtained by adding methyl lithium (19 ml of a 1.6 M solution in ether, 30 mmol) to a mixed suspension of copper iodide (2.86 g, 15 mmol) in ether (30 ml) at 0 ° C in an inert atmosphere argon. After stirring for 15 minutes in the cold, a solution of C-105 tetraene (1.6 g, 15 mmol) in tetrahydrofuran was added dropwise over 25 minutes. The reaction was continued for another 30 minutes and the reaction mixture was poured into a saturated solution of ammonium chloride with vigorous stirring. The aqueous mixture was extracted with ethyl acetate and methylene chloride. The combined organic layers were washed with an aqueous solution of ammonium chloride, water and dried with sodium sulfate. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate or methylene chloride and treated with p-toluenesulfonic acid (100 mg) in a steam bath for a period of time from 30 minutes to 1 hour. The organic solution was washed with water and dried with sodium sulfate. The solvent was removed in vacuo to give crude C-119 enon. Purification was carried out by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane. Alternatively, the crude C-119 enon was chromatographed on silica gel, after which the product was recrystallized. See, for example, the procedure described in J.K. Grunwell et al., Steroids, 27, 759-771 (1976), which is incorporated herein by reference.

Example X-19. Receiving:

S-120 ester (4.00 g, 10 mmol):

(obtained in accordance with the procedure described in RMWeier & LMHofmann, J. Med. Chem., 18, 817 (1975), which is incorporated herein by reference) were reacted with trifluoroacetic acid (25 ml), trifluoroacetic anhydride acid (4.5 ml) and potassium acetate (6.7 g, 7.1 mmol) in accordance with the procedure described for the synthesis of Compound C-1 in Example X-1. Crude product C-121 was isolated according to the procedure described for the synthesis of Compound C-1 in Example X-1, and purified by silica gel chromatography using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents, and the product thus obtained was additionally purified by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane.

Example X-20. Receiving:

Enon S-122 (3.68 g, 10 mmol):

(obtained in accordance with the procedure described in US patent 3499891, issued 03/10/1970 FBColton & RTNicholson, which is introduced into the present description by reference) were reacted with potassium acetate (6.7 g, 7.1 mmol), trifluoroacetic acid (25 ml) and trifluoroacetic acid anhydride (4.5 ml) in accordance with the procedure described for the synthesis of Compound C-1 in Example X-1. The crude product was isolated according to the above procedure and purified by silica gel chromatography using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents, and the product thus obtained was further purified by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane.

Example X-21. Receiving:

and

Compounds C-125 and C-126 were synthesized in accordance with the procedure used above for the synthesis of Compounds C-108 and C-1. To a solution of trimethyl sulfoxonium iodide (1 g, 4.6 mmol) in dry dimethyl sulfoxide (20 ml) was added sodium hydride (220 mg of a 50% dispersion in mineral oil, 4.6 mmol). The mixture was stirred at room temperature under a nitrogen atmosphere until hydrogen evolution ceased.

Then was added a solution of trienone C-124 (1.14 g, 3.5 mmol):

in dimethyl sulfoxide (4 ml) and stirring was continued for 4 hours under nitrogen. Trienone C-124 was obtained in accordance with the procedure described in Example X-2 to obtain tetraene C-105, using canrenone instead of 11α-hydroxy-canrenone as the starting compound. The reaction mixture was diluted with water and the resulting precipitate was filtered and dried in air. The product was a mixture of 6β, 7β- (Compound C-125) and 6α, 7α- (Compound C-126) isomers. The separation of these isomers was carried out by silica gel chromatography and the individual isomers were further purified by recrystallization from solvents such as ethyl acetate and hexane, alcohol or alcohol and water.

Example X-22. Preparation of methyl 3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norandrosta-1,4,8,14-tetraen-7α-carboxylate ( Compounds C-127)

Dienone C-127 was synthesized in accordance with the procedure described in R.M. Weier & L.M. Hofmann, J.Med.Chem., 18, 817 (1975), which is incorporated herein by reference. A solution of Compound C-1 (3.8 g, 10 mmol) and dichlorodicyanobenzoquinone (2.72 g, 12 mmol) in dioxane (80 ml) was heated under stirring under reflux for 24 hours. The reaction mixture was concentrated in vacuo, the residue was hydrolyzed with methylene chloride, filtered and the filtrate was washed with 2% sodium sulfite, 5% sodium hydroxide and saturated sodium chloride solution, and then dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo. The crude C-127 dienone product was purified by silica gel chromatography using mixtures of ethyl acetate and toluene as eluents, and the product 27 thus obtained was further purified by recrystallization from alcohol.

Example X-23. Preparation of 2 ', 3', 3'aα, 4 ', 6', 11'aα, 12 ', 13'-octahydro-3'aR, 3'a, 11'a-dimethyl-13'aR * -spiro- [cyclopropane-1.7 '(9'H) - [1H] pentaleno- [1,6a-a] phenanthrene-1'9'-dione (Compounds C-128)

Using the procedure and processing described in Example X-22 above for the synthesis of C-127 dienone, C-114 enone (3.48 g, 10 mmol) was converted to C-128 dienone using dichlorodicyanobenzochione (2.72 g, 12 mmol) ) in dioxane (80 ml).

Example X-24. Preparation of 4bR (4bR *), 5.5aS *, 7.7aR *, 8.9.11, -12.12bS * -decahydro-7A, 12b-dimethyl-10R * -cycloprop (1) pentaleno [1,6- a] phenanthrene-3,10-dione (Compounds C-129)

Using the procedure and processing described in Example X-22 above for the synthesis of C-127 dienone, C-108 enone (3.34 g, 10 mmol) was converted to C-129 dienone using dichlorodicyanobenzoquinone (2.72 g, 12 mmol) ) in dioxane (80 ml).

Example X-25. Receiving:

To a cold (0 ° C) stirred solution of C-101 carboxylic acid (3.66 g, 10 mmol) and N-methylmorpholine (1.01 g, 10 mmol) in tetrahydrofuran (35 ml) was added isobutyl chloroformate (1.36 g, 10 mmol). The reaction mixture was stirred for 20 minutes at 0 ° C, filtered and the filtrate was concentrated in vacuo. The residue was mixed anhydride and was suitable for use in the next step.

Dimethylamine gas was bubbled into a cold (0 ° C) solution of mixed anhydride (4.6 g, 10 mmol) in tetrahydrofuran (40 ml) in a pressure vessel. After 15 minutes, this pressure vessel was sealed and the reaction mixture was left for 24 hours at room temperature. The reaction mixture was heated to 40 ° C for 30 minutes, and then cooled again to 0 ° C. After warming to room temperature, the pressure in the vessel was reduced to atmospheric and left to evaporate the excess dimethylamine. The reaction mixture was concentrated in vacuo, the residue was dissolved in ethyl acetate and extracted with 1N sodium hydroxide and water. After drying with sodium sulfate, the organic layer was evaporated and the residue was purified by silica gel chromatography using mixtures of ethyl acetate and toluene as eluents. The C-130 amide thus obtained was isolated and further purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane.

Example X-26. Obtaining a compound of the formula:

To a suspension of enone C-114 (3.5 g, 10 mmol) in ethyl orthoformate (10 ml) and anhydrous ethanol (10 ml) was added p-toluenesulfonic acid monohydrate (0.05 g). The reaction mixture was stirred for 30 minutes at room temperature and quenched by adding a few drops of pyridine. After stirring for another 5 minutes at 0 ° C, the resulting precipitate was filtered, washed with a small amount of methanol and recrystallized from alcohol or ethyl acetate and hexane containing trace amounts of pyridine, whereby pure C-131 enol ether was obtained. Alternatively, after pyridine is added, this reaction mixture can be treated by removing all solvents in vacuo and crystallizing the residue by adding solvents such as hexane, ether, ethyl acetate or hexane. The crude enol ether C-131 was recrystallized as described above.

Example X-27. Receiving:

To a cooled (-78 ° C) solution of lithium bis (trimethylsilyl) amide (10 ml of a 1.0 M solution in tetrahydrofuran) was added dropwise a solution of C-131 enol ether (3.8 g, 10 mmol) in tetrahydrofuran (20 ml). The reaction mixture was stirred at -78 ° C for 10 minutes and a solution of phenylselenyl chloride (1.9 g, 10 mmol) in tetrahydrofuran (5 ml) was added. The reaction mixture was stirred for 5 minutes and quenched by adding 1% sodium bisulfate solution. Then the reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with 5% aqueous sodium bicarbonate and water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents to give pure C-132 selenide.

Example X-28. Receiving:

To a cooled (0 ° С) solution of С-132 selenide (g, 10 mmol) and pyridine (I, 61 ml, 20 mmol) in methylene chloride (40 ml), a solution of hydrogen peroxide (3.1 g of a 30% solution, 27 mmol) in water (3 ml). The temperature was maintained at less than 30-35 ° C. After the exotherm decreased, the water bath was removed and the reaction mixture was vigorously stirred for 15 minutes at room temperature. The reaction mixture was diluted with methylene chloride and washed with 5% sodium bicarbonate solution and water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents to give pure C-133 unsaturated ketone, which was then purified by recrystallization from ethyl acetate and hexane or alcohol.

Example X-29. Receiving:

A solution of K-133 ketone (2 g) in acetone (15 ml) was treated with 1N hydrochloric acid (4 ml) for 1 hour at room temperature. The reaction mixture was concentrated in vacuo. The residue was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with 5% sodium bicarbonate solution and water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuo to give crude C-134 ketone. This crude product was purified by silica gel chromatography using mixtures of ethyl acetate and toluene as eluents to give pure C-134 ketone, which was further purified by recrystallization from ethyl acetate and hexane or alcohol.

Claims (58)

1. The method of obtaining 3-keto-7α-alkoxycarbonyl-substituted Δ4,5-steroid of the General formula

Where

selected from

and

R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ²¹ represents hydrogen or alkyl; R ²⁶ represents C ₁ -C ₄ alkyl; R ⁸ and R ⁹ taken together form a heterocyclic ring system, comprising the interaction of an alkylating agent with a 4,5-dihydro-5,7-lactone steroid substrate of the general formula:

where R ¹⁸ represents a C ₁ -C ₄ alkyl, or R ¹⁸ O-groups taken together form an O, O-hydroxyalkylene bridge or a keto group, and

, R ³ , R ⁸ and R ⁹ are as defined above in the presence of a base. 2. The method according to claim 1, where the specified steroid substrate and the specified steroid product are substituted at the 17-position with a spirolactone substituent corresponding to the formula XXXIII

3. The method according to claim 1, where the specified lactone substrate contains 3-dialkoxy substituents. 4. The method according to claim 1, where the specified 5,7-lactone is reacted with an alkyl halide in the presence of a base. 5. A method of obtaining a 4,5-dihydro-5,7-lactone steroid derivative of the formula

Where

, R ³ , R ⁸ , R ⁹ and R ¹⁸ have the meanings where indicated in claim 1, which consists in converting the corresponding 7-cyano-substituted steroid to a 7-carboxy-substituted steroid and then converting the 7-carboxy-substituted steroid into the corresponding 5,7-lactone-substituted steroid . 6. The method according to claim 5, where in the Δ-4,5-7-carboxy-substituted steroid of group OR ¹⁸ taken together form a 3-keto group, which is converted to obtain a ketal intermediate containing 3-dialkoxy-5,7-lactone and said 3-dialkoxy-5,7-lactone is then hydrolyzed under acidic conditions to give 3-keto-5,7-lactone. 7. A method of obtaining a compound corresponding to formula II

, where R ¹ represents an alpha oriented C ₁ -C ₄ alkoxycarbonyl radical; R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸ and R ⁹ taken together form a heterocyclic ring system; which comprises alkylating a compound of formula EI by hydrolysis followed by reaction with an alkylating reagent in the presence of a base, where the compound of formula EI has the structure:

, where R ³ , R ⁸ and R ⁹ have the meanings indicated above; R ¹⁷ represents C ₁ -C ₄ alkyl. 8. The method of obtaining the compounds of formula IIC

, where R ¹ represents an alpha oriented C ₁ -C ₄ alkoxycarbonyl or hydroxycarbonyl radical; R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; which comprises alkylating a compound of general formula E by hydrolysis followed by reaction with an alkylating reagent in the presence of a base, where the compound of formula E has the structure

, where R ³ has the meanings indicated above; R ¹⁷ represents C ₁ -C ₄ alkyl. 9. The method of obtaining the compounds of formula E

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁷ represents C ₁ -C ₄ alkyl; which includes the thermal decomposition of a compound corresponding to the formula DE2 in the presence of an alkali metal halide, wherein said compound of the formula DE2 has the structure

, where R ¹² represents C ₁ -C ₄ alkyl; R ³ and R ¹⁷ have the meanings indicated above. 10. The method of obtaining the compounds of formula DE2

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹² and R ¹⁷ are C ₁ -C ₄ alkyl; which comprises condensing a compound of formula DE1 with dialkyl malonate in the presence of a base, wherein said compound of formula DE1 has the structure:

, where R ³ and R ¹⁷ have the meanings indicated above. 11. The method of obtaining the compounds of formula DE1

, where R ³ selected from the group comprising hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁷ represents C ₁ -C ₄ alkyl; which comprises reacting a compound of formula D with a sulfonium ylide in the presence of a base, wherein said compound of formula D has the structure:

, where R ³ and R ¹⁷ have the meanings indicated above. 12. The method of obtaining the compounds of formula D

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁷ represents C ₁ -C ₄ alkyl; which comprises hydrolysis of a compound of formula C to form 7α-carboxylic acid and reacting under acidic conditions with a trialkyl orthoformate, wherein the compound of formula C has the structure

, where R ³ has the meanings indicated above. 13. The method of obtaining the compounds of formula I

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ²⁶ represents C ₁ -C ₄ alkyl; R ⁸⁰ and R ^{90 are} independently selected from R ⁸ and R ^9, respectively, or R ⁸⁰ and R ⁹⁰ together form a keto group; which comprises alkylating a compound of formula A211 by reaction with an alkylating reagent in the presence of a base, where the compound of formula A211 has the structure

, where R ³ , R ⁸⁰ and R ⁹⁰ have the meanings indicated above. 14. The method of obtaining the compounds of formula IE

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ²⁶ represents C ₁ -C ₄ alkyl; which comprises alkylating a compound of general formula 211 by reaction with an alkylating reagent in the presence of a base, where the compound of formula 211 has the structure

, where R ³ has the meanings indicated above. 15. The method of obtaining the compounds of formula A211

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸⁰ and R ^{90 are} independently selected from R ⁸ and R ^9, respectively, or R ⁸⁰ and R ⁹⁰ together form a keto group; R ⁸ and R ⁹ independently represent hydroxycarbonylalkyl, or R ⁸ and R ⁹ together represent a heterocyclic ring structure; which comprises oxidizing a compound of formula A210, wherein the compound of formula A210 has the structure

, where R ³ , R ⁸⁰ and R ⁹⁰ have the meanings indicated above. 16. The method according to clause 15, where R ⁸⁰ and R ⁹⁰ together with C (17) denote

where X is oxo; Y ¹ represents hydroxy; Y ² represents hydroxy or lower alkoxy. 17. The method according to clause 15, where R ⁸⁰ and R ⁹⁰ taken together with C (17), denote

18. The method of obtaining the compounds of General formula A211

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸ and R ⁹ independently represent hydroxycarbonylalkyl, or R ⁸ and R ⁹ taken together represent a heterocyclic ring structure; which comprises reacting an intermediate 3-keto-5,7-half-acetal of formula A209C with a peroxide oxidizing agent, wherein said compound of formula A209C corresponds to the formula

where R ³ , R ⁸ and R ⁹ have the meanings indicated above. 19. The method according to p. 18, where R ⁸ and R ⁹ taken together with C (17), denote

where X is oxo; Y ¹ represents hydroxy; Y ² represents hydroxy or lower alkoxy. 20. The method according to p. 18, where R ⁸ and R ⁹ taken together with C (17), denote

21. A method of obtaining a compound of General formula A210

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸⁰ and R ^{90 are} independently selected from R ⁸ and R ^9, respectively, or R ⁸⁰ and R ⁹⁰ together form a keto group; R ⁸ and R ⁹ independently represent hydroxycarbonylalkyl, or R ⁸ and R ⁹ together represent a heterocyclic ring structure; which comprises reacting an intermediate 3-keto-5,7-half-acetal of formula A209C with a peroxide oxidizing agent, wherein said compound of formula A209C corresponds to the formula

where R ³ , R ⁸ and R ⁹ have the meanings indicated above. 22. The method according to item 21, where R ⁸ and R ⁹ taken together with C (17), denote

where X is oxo; Y ¹ represents hydroxy; Y ² represents hydroxy or lower alkoxy. 23. The method according to item 21, where R ⁸ and R ⁹ taken together with C (17), denote

24. A method of obtaining a compound corresponding to the formula A209,

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸⁰ and R ^{90 are} independently selected from R ⁸ and R ^9, respectively, or R ⁸⁰ and R ⁹⁰ together form a keto group; R ⁸ and R ⁹ independently represent hydroxycarbonylalkyl or R ⁸ and R ⁹ together represent a heterocyclic ring structure; -E-E- selected from the group including

and

where R ²¹ represents hydrogen or alkyl; which includes hydrolysis of a compound of general formula A208

, where -E-E-, R ³ , R ⁸⁰ and R ⁹⁰ have the meanings indicated above; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; R ²⁰ represents C ₁ -C ₄ alkyl. 25. A method of obtaining a compound of General formula A205;

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; R ²⁰ represents C ₁ -C ₄ alkyl; -E-E- selected from the group including

and

where R ^{21 is} independently selected from hydrogen and alkyl; which comprises reacting a compound of general formula A204 with a lower alcohol and an acid, wherein said compound of formula A204 has the structure

, where -E-E-, R ³ and R ¹⁹ have the meanings indicated above. 26. A method of obtaining a compound of formula A204

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; -E-E- selected from the group including

,

and

where R ^{21 is} independently selected from hydrogen and alkyl; which includes hydrolysis of a compound of general formula A203, wherein said compound of formula A203 has the formula

where -E-E- and R ³ have the meanings indicated above; R ¹⁸ represents C ₁ -C ₄ alkyl, or R ¹⁸ O - groups taken together form an O, O-hydroxyalkylene bridge. 27. A method of obtaining a compound of formula A204

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; -E-E- selected from the group including

and

where R ^{21 is} independently selected from hydrogen and alkyl; which includes protecting keto substituents of the compounds of formula A201 by reacting with alkanol under acidic conditions in the presence of orthoformate, wherein said compound of formula A201 has the structure

, where -E-E- and R ³ have the meanings indicated above; thereby obtaining the 3-enol ether intermediate of formula A202

, where -E-E- and R ³ have the meanings indicated above; R ¹⁸ represents C ₁ -C ₄ alkyl, or R ¹⁸ O groups taken together form an O, O-hydroxyalkylene bridge; recovering said compound of formula A202 to obtain a compound of formula A203:

, where -E-E-, R ³ and R ¹⁸ have the meanings indicated above; hydrolysis of said compound of formula A203 to thereby give a compound of formula A204. 28. A method of obtaining a compound of formula A203:

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁸ represents C ₁ -C ₄ alkyl, or R ¹⁸ O-groups at C-17, taken together, form a 0,0-hydroxyalkylene bridge; -E-E- selected from the group including

and

where R ^{21 is} independently selected from hydrogen and alkyl; which includes the recovery of the compounds of formula A202:

where -E-E-, R ³ and R ¹⁸ have the meanings indicated above. 29. The method according to any one of claims 8 to 28, where R ³ represents hydrogen. 30. The method according to any one of paragraphs.24-28, where R ³ represents hydrogen and -E-E- selected from

and

31. A method of producing an epoxy compound, comprising contacting a substrate compound having an olefinic double bond with a peroxide compound in the presence of a peroxide activator, wherein said peroxide activator is chlorodifluoroacetamide or corresponds to a compound of the formula

, Where R ^{p is} selected from the group consisting of alkenyl, alkynyl and - (CX ⁴ X ⁵ ) _n -; X ¹ , X ² , X ³ , X ⁴ and X ^{5 are} independently selected from halogen, hydrogen, alkyl, haloalkyl, cyano and cyanoalkyl; n is 0, 1 or 2; provided that when n is 0, then at least one of X ¹ , X ² and X ³ is halogen, when R ^p represents - (CX ⁴ X ⁵ ) _n - and n is 1 or 2, then at least one of X ⁴ and X ⁵ is halogen. 32. The method according to p, where n is 0 and at least two of X ¹ , X ² and X ³ are halogen or perhalogenated. 33. The method according to p, where all X ¹ , X ² , X ³ , X ⁴ and X ⁵ represent halogen or perhalogenated. 34. The method of claim 31, wherein said peroxide activator is selected from the group consisting of chlorodifluoroacetamide and heptafluorobutyramide. 35. The method according to p, where the specified substrate compound has the formula II

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹ represents an alpha oriented C _1-4 alkoxycarbonyl radical; R ⁸ and R ^{9 are} independently selected from hydroxycarbonylalkyl; or R ⁸ and R ⁹ taken together form a heterocyclic ring structure. 36. The method according to clause 35, where R ³ represents hydrogen. 37. The method according to p, where the specified substrate compound is selected from the group including

and the product of the epoxidation reaction is selected from the group including

38. The method according to p, where the specified substrate compound is selected from the group including

and the product of the epoxidation reaction is selected from the group including

39. The compound of formula D

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁷ represents C ₁ -C ₄ alkyl. 40. The compound of formula E

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁷ represents C ₁ -C ₄ alkyl. 41. The compound of formula F

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy and cyano. 42. The compound of formula 211

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy and cyano. 43. The compound of formula A210

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸⁰ and R ^{90 are} independently selected from R ⁸ and R ^9, respectively, or R ⁸⁰ and R ⁹⁰ together form a keto group; R ⁸ and R ^{9 are} independently selected from hydroxycarbonylalkyl; or R ⁸ and R ⁹ taken together form a heterocyclic ring structure. 44. The compound of formula A209

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ⁸⁰ and R ^{90 are} independently selected from R ⁸ and R ^9, respectively, or R ⁸⁰ and R ⁹⁰ together form a keto group; R ⁸ and R ^{9 are} independently selected from hydroxycarbonylalkyl, or R ⁸ and R ⁹ taken together form a heterocyclic ring structure; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 45. The compound of formula 208:

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; R ²⁰ represents C ₁ -C ₄ alkyl; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 46. The compound of formula 207

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; R ²⁰ represents C ₁ -C ₄ alkyl; R ²⁵ represents C ₁ -C ₄ alkyl; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 47. The compound of formula A206

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; R ²⁰ represents C ₁ -C ₄ alkyl; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 48. The compound of formula A205

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; R ²⁰ represents C ₁ -C ₄ alkyl; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 49. The compound of formula A204

, where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁹ represents C ₁ -C ₄ alkyl, or R ¹⁹ O groups taken together form an O, O-hydroxyalkylene bridge; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 50. The compound of formula A203

where R ³ represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano; R ¹⁸ represents C ₁ -C ₄ alkyl, or R ¹⁸ O-groups at C-17, taken together, form an O, O-hydroxyalkylene bridge; -E-E- selected from

and

where R ²¹ represents hydrogen or alkyl. 51. The compound of claim 50 of formula A203, wherein R ³ is hydrogen. 52. The compound according to any one of paragraphs.44-51, where R ³ represents hydrogen; -E-E- selected from

and

where R ²¹ represents hydrogen. 53. The compound according to any one of paragraphs.44-51, where R ³ represents hydrogen; -E-E- represents

where R ²¹ represents hydrogen. 54. The compound of formula 303 where R ¹ represents an alpha oriented C ₁ -C ₄ alkoxycarbonyl or hydroxycarbonyl radical; R ⁸ and R ⁹ independently represent hydroxycarbonylalkyl, or R ⁸ and R ⁹ together represent a heterocyclic ring structure. 55. The compound of claim 54 of formula 303A

where R ¹ represents an alpha oriented C ₁ -C ₄ alkoxycarbonyl or hydroxycarbonyl radical; X is oxo; Y ¹ represents hydroxy; Y ² represents hydroxy or lower alkoxy. 56. The compound of claim 55 of the formula

Priority on points: 12/11/1996 according to claims 1-30, 39-56; 12/11/1997 according to paragraphs 31-38.

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