Method for preparing 3-keto-7α-alkoxycarbonyl-subtituted Δ4,5-steroid, methods for preparing intermediate compounds, intermediate compounds

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

 

Background of invention

The present invention relates to new methods of obtaining 9,11-amoxiciloin derivatives, in particular compounds of the number of 20-spirostanol and their analogues; to new intermediate compounds used for the production of steroidal compounds; and to methods of producing these new intermediates. More specifically, the invention relates to new and improved methods of producing Metelitza 9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone (also known as eplerenone or aproximaciones) (onomatology ether γ-lactone 9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-dicarboxylic acid).

Methods for obtaining compounds of the number of 20-spirostanol described in U.S. patent No. 4559332. Compounds produced by the method described in patent 4559332 have an open oxygen-containing ring E. the General formula:

where

-A-a - represents the group-CH2-CH2- or-CH=CH-;

R1is α-oriented lower alkoxycarbonyl or hydroxycarbonyl radical;

-B-B - represents the group-CH2-CH2or αor β-oriented group;

R6and R7represent hydrogen;

X submitted is two hydrogen atoms or oxo;

Y1and Y2together represent the oxygen bridge-O-, or

Y1represents hydroxy, and

Y2represents hydroxy, lower alkoxy or, if X represents H2also lower alkanoyloxy;

and salts of such compounds, in which X represents oxo and Y2represents hydroxy, i.e. salts of the corresponding 17β-hydroxy-21-carboxylic acid.

In U.S. patent No. 4559332 describes several methods of obtaining epoxyoctane and close to the structure of the compounds of formula IA. The emergence of new and wider clinical applications epoxyoctane leads to the need to improve ways of getting it and the other close to the structure of steroids.

Brief description of the invention

The main aim of the present invention is to develop improved methods of obtaining epoxyoctane, other 20-spirostanol and other steroids having the General structural characteristics. Some of the specific objectives of the present invention are: to develop improved ways to obtain products of formula IA and other closely related structures of compounds with high yield; to provide such a method which provides for the implementation of the minimum stages of selection; and to provide such a method which can be implemented with reasonable capital costs and reasonable the cost, associated with the conversion.

Accordingly, the present invention relates to a number of schemes for the synthesis epoxyoctane; intermediates which can be used to produce epoxyoctane; and to the synthesis of these new intermediate compounds.

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

The compound of formula IV corresponds to the structure

where:

-A-a - represents the group-CHR4-CHR5or CR4=CR5-;

R3, R4and R5independently selected from the group comprising hydrogen, halogen, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy;

R1represents an alpha-oriented lower alkoxy-carbonyl or hydroxycarbonyl radical;

R2is 11α-remove the group, the cleavage of which is effective for the introduction of a double bond between the 9 - and 11-carbon atoms;

- - Represents a group-CHR6-CHR7-or alpha - or beta-oriented group:

where R6and R7independently selected from the group consisting of bodoro is, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, aryloxyalkyl, cyano, aryloxy; and

R8and R9independently selected from the group comprising hydrogen, hydroxy, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, aryloxyalkyl, cyano, aryloxy; or R8and R9together form a carbocyclic or heterocyclic ring structure, or R8or R9together with R6or R7form a carbocyclic or heterocyclic ring structure fused with Pyh ring D.

The compound of formula IVA corresponds to the formula IV, where R8and R9together with the ring carbon atom to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula IVB corresponds to the formula IV, where R8and R9together form the structure of formula XXXIII:

Compounds of formula IVC, IVD and IVE, respectively, correspond to any of formulas IV, IVA or IVB, where each of the-And-And - and-In-are-CH2-CH2-, R3is hydrogen and R1is alkoxycarbonyl, preferably, methoxycarbonyl. Connections covered forms the Loy IV, can be obtained by reacting a lower-alkylsulfonamides or Alliluyeva reagent or halide-forming agent with the appropriate compound of formula V.

The compound of formula V corresponds to the structure:

where: -a-a -, - - -, R1, R3, R8and R9defined in formula IV.

The compound of formula VA corresponds to the formula V, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula VB corresponds to the formula V, where R8and R9together form the structure of formula XXXIII:

Compounds of formula VC, VD and VE, respectively, correspond to any one of formulas V, VA or VB, where each of the-And-And - and- -represents-CH2-CH2-, R3is hydrogen and R1is alkoxycarbonyl, preferably methoxycarbonyl. Compounds covered by the formula V can be obtained by reacting the alkoxide of an alkali metal with an appropriate compound of formula VI.

The compound of formula VI corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined in formula IV.

The compound of formula VA corresponds to the formula VI, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula VIB corresponds to the formula VI, where R8and R9together form the structure of formula XXXIII:

Compounds of formula VIC, VID and VIE, respectively, correspond to any of formula VI, VIA or VIB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds of formula VI, VIA, VIB and VIC are obtained by hydrolysis of compounds corresponding to formula VII, VIIA, VIIB or VIIC, respectively.

Compound of formula VII corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined above for formula IV.

The compound of formula VIIA corresponds to formula VII, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

Compound of formula VII corresponds to the formula VII, where R8and R9together form the structure of formula XXXIII:

Compounds of formula VIIC, VIID and VIIE, respectively, correspond to any of formula VII, VIIA, or VIIB, where each of the-AA and- - - represents-CH 2-CH2-, and R3represents hydrogen. The connection is covered by formula VII can be obtained by zenderoudi compounds covered by formula VIII.

The compound of formula VIII corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined above in formula IV.

The compound of formula VIIIA corresponds to the formula VIII, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula VIII corresponds to the formula VIII, where R8and R9together form the structure of formula XXXIII:

Compounds of formula VIIIC, VIIID and VIIIE, respectively, correspond to any of formula VIII, VIIIA or VIIIB, where each one-And-A - and-B-B - represents-CH2-CH2and R3represents hydrogen. Compounds covered by formula VIII are obtained by oxidation of the substrate containing the compound of formula XXX, as described below, by fermentation, carried out for the introduction of 11-hydroxy-group in the substrate in α-orientation. The compound of formula IX corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined above for formula IV, and R1set the n to formula V.

The compound of formula IXA corresponds to formula IX, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula IXB corresponds to formula IX, where R8and R9together with the ring carbon to which they are attached, form a structure of formula XXXIII:

Compounds of formula IXC, IXD and IXE, respectively, correspond to any of formula IX, IXA, or IXB, where each one-And-A - and-B-B - represents-CH2-CH2and R3represents hydrogen. Compounds covered by formula IX can be obtained by conversion of the corresponding compounds of formula X.

The compound of formula XIV corresponds to the structure;

where: -a-a -, - - -, R3, R8and R9defined above in formula IV.

The compound of formula XIVA corresponds to the formula XIV, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula XIVB corresponds to the formula XIV, where R8and R9together with the ring carbon to which they are attached, form a structure of formula XXXIII:

Compounds of formula XIVC, XIVD and XIVE, respectively, correspond to any of formula XIV, XIVA or XIVB, where each one-And-A - and-B-B - represents-CH2-CH2and R3represents hydrogen. Compounds covered by formula XIV can be obtained by hydrolysis of the corresponding compounds of formula XV.

The compound of formula XV corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined above for formula IV.

The compound of formula XVA corresponds to the formula XV, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula XVB corresponds to the formula XV, where R8and R9together with the ring carbon to which they are attached, form a structure of formula XXXIII:

Compounds of formula XVC, XVD and XVE, respectively, correspond to any of formula XV, XVA, or XVB, where each one-And-A - and-B-B - represents-CH2-CH2and R3represents hydrogen. Compounds covered by formula XV can be obtained by zenderoudi corresponding compounds covered by formula XVI.

The compound of formula XXI corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined above for formula IV.

The compound of formula XXIA corresponds to the formula XXI, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula XXIB corresponds to the formula XXI, where R8and R9together form the structure of formula XXXIII:

Compounds of formula XXIC, XXID and XXIE, respectively, correspond to any of formula XXI, XXIA or XXIB, where each of the-And-And - and-In-To - represent-CH2-CH2-, and R3represents hydrogen. Compounds covered by the 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 -, - - -, R3, R8and R9defined above for formula IV.

The compound of formula XXIIA corresponds to the formula XXII, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula XXIIB corresponds to the formula XXII, where R8and R9together form the structure of formula XXXIII:

Compounds of formula XXIIC, XXIID and XXIIE, respectively, correspond to any of formula XXII, XXIIA or XXIIB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by formula XXII can be obtained by zenderoudi the corresponding compounds of formula XXIII.

The compound of formula XXIII corresponds to the structure:

where: -a-a -, - - -, R3, R8and R9defined above for formula IV.

The compound of formula XXIIIA corresponds to the formula XXIII, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) defined above.

The compound of formula XXIIIB corresponds to the formula XXIII, where R8and R9together form the structure of formula XXXIII:

Compounds of formula XXIIIC, XXIIID and XXIIIE, respectively, correspond to any of formula XXIII, XXIIIA or XXIIIB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula XXIII can be obtained by oxidation of compounds of formula XXIV, as described below.

The compound of formula XXVI corresponds to the structure:

where: -a-a -, - - -, R3, R8and R 9defined above for formula IV.

The compound of formula XXVIA corresponds to the formula XXVI where every one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula XXVI can be obtained by oxidation of compounds of formula XXVII.

The compound of formula XXV corresponds to the structure:

where; -a-a -, - - -, R3, R8and R9defined above for formula IV.

The compound of formula XXVA corresponds to the formula XXV, where each one-And-A - and-B-B - represents the group-CH2-CH2-, and R3represents hydrogen. Compounds covered by formula XXV can be obtained by zenderoudi the compounds of formula XXVI.

The compound of formula 104 corresponds to the structure:

where: -a-a-, -B-b - and R3defined above for formula IV, and R11is1-C4-alkyl.

The compound of formula I corresponds to the formula 104, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by formula 104, can be obtained by thermal decomposition of compounds of formula 103.

The compound of formula 103 corresponds to the structure:

where: -a-a -, - - -, R3and R11defined above for formula 104, and R1 is1-C4-alkyl.

The compound of formula 103A corresponds to the formula 103, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by formula 103, can be obtained by reacting the corresponding compounds of formula 102 with diallylmalonate in the presence of a base such as an alkali metal alkoxide.

The compound of formula 102 corresponds to the structure;

where: -a-a -, - - -, R3and R11defined above for formula 104.

The compound of formula 102A corresponds to the formula 102, where each of the-a-a - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by formula 102, can be obtained by reacting the corresponding compounds of formula 101 with trialkylaluminium compound in the presence of a base.

The compound of formula 101 corresponds to the structure:

where: -a-a -, - - -, R3and R11defined above for formula 104.

The compound of formula 101A corresponds to the formula 101, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by formula 101, can be obtained by reacting 11α-hydroxyandrost-3,17-dione or another connected to the I formula XXXVI with trialkyl-ortho-formate in the presence of acid.

The compound of formula XL corresponds to the formula;

where-e-E - is selected from:

and

R21, R22and R23independently selected from hydrogen, alkyl, halogen, nitro and cyano; R24selected from hydrogen and lower alkyl; R80and R90independently selected from keto and substituents, which may be R8and R9(as defined above in relation to formula (IV) ; and-And-And-In-In - and R3defined for formula IV.

The compound of formula XLA corresponds to the formula XL, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

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

The compound of formula XLE corresponds to the formula XL, where R80and R90together with the ring carbon to which they are attached, represent a keto or:

where X, Y1, Y2and(17) defined above, or

The compounds of formula XLIE correspond to the formula XL, where R80and R90together form keto.

Compounds of formula XLF, XLG, XLH, XLJ, XLM and XLN correspond to the formula XL, XLA, XLB, XLC, XLD and XLE, respectively, in which-a-a-, -B-b - and R3defined above.

The compound of formula XLI corresponds to the formula:

where-e-E - is selected from:

and

R18represents C1-C4-alkyl, or the group R18O together form an O,O-oxyalkylene bridge; R21, R22and R23independently selected from hydrogen, alkyl, halogen, nitro and cyano; R24selected from hydrogen and lower alkyl; R80and R90independently selected from keto and substituents, which may be R8and R9; and-And-And-In-In - and R3defined in formula IV.

The compound of formula XLIA corresponds to the formula XLI, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

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

The compound of formula XLIC corresponds to the formula XLI, where R80and R90together with the ring carbon to which they are attached, represent a keto or:

where: X, Y1, Y2and(17) defined above.

The compounds of formula XLID correspond to the formula XLI, where Deputy XXXIV corresponds to the structure XXXIII

The compounds of formula XLIE correspond to the formula XL, where R80and R90together form keto.

Compounds of formula XLIF, XLIG, XLIH, XLIJ, XLIM and XLIN correspond to the formula XLI, XLIA, XLIB, XLIC, XLID and XLIE, respectively, where-a-a-, -B-b - and R3defined above. Compounds covered by the formula XLI, obtained by hydrolysis of the corresponding compounds of formula XL are shown below.

The compound of formula XLII corresponds to the formula:

where-e-E - is selected from:

and

R21, R22and R23independently selected from hydrogen, alkyl, halogen, nitro and cyano; R24selected from hydrogen and lower alkyl; R80and R90independently selected from keto and substituents, which may be R8and R9and-And-And-In-In - and R3defined in formula IV.

The compound of formula XLIIA correspond to the formula XLII, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

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

The compound of formula XLIIC corresponds to the formula XLII, where R80and R90together with the ring carbon to which they are attached, represent a keto or:

where X, Y1, Y2and(17) defined above.

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

The compounds of formula XLIIE correspond to the formula XLII, where R80and R90together form keto. Compounds of formula XLIIF, XLIIG, XLIIH, XLIIJ, XLIIM and XLIIN correspond to the formula XLII, XLIIA, XLIIB, XLIIC, XLIID and XLIIE, respectively, where-a-a - and-B-To - represent-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula XLII, obtained by removal of protection from the corresponding compounds of formula XLI.

The compound of formula XLIX is similar to the structure:

where-e-E - defined for formula XL, a-a-a -, - - -, R1, R3, R8and R9defined in formula IV.

The compound of formula XLIXA corresponds to the formula XLIX, where R8and R9together with the ring carbon to which they are attached form the structure:

where X, Y1, Y2and(17) determine the s above.

The compound of formula XLIXB corresponds to the formula XLIX, where R8and R9together form the structure of formula XXXIII:

Compounds of formula XLIXC, XLIXD, XLIXE, respectively, correspond to any of formula XLIX, XLIXA or XLIXB, where each one-And-A - and-B-B - represents-CH2-CH2-, R3represents hydrogen, and R1is alkoxycarbonyl, preferably methoxycarbonyl. Compounds covered by formula XLIX can be obtained by reacting an alcohol or an aqueous solvent with an appropriate compound of formula VI in the presence of a suitable base.

The compound of formula A corresponds to the structure:

where-e-E - is selected from:

and

R18selected from C1-C4-alkyl; R21, R22and R23independently selected from hydrogen, alkyl, halogen, nitro and cyano; R24selected from hydrogen and lower alkyl; a-a-a-, -B-b - and R3defined for formula IV.

The compound of formula AA corresponds to the formula A, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula AA where-is-S - corresponds to the formula XLIII, XLIV, XLV or XLVII.

Compounds of formula IS, A203D and AE, respectively, correspond to the formula A, AA and AB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula A, obtained by recovery of the compounds of formula A below.

The compound of formula A corresponds to the structure:

where R19is1-C4-alkyl and-S-E-, -a-a-, -B-b - and R3defined for formula 203.

The compound of formula AA corresponds to the formula A, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula IA, where-e-E - corresponds to the formula XLIII, XLIV, XLV or XLVII.

Compounds of formula IS, A204D and AE, respectively, correspond to the formula A, AA and AB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula A, obtained by hydrolysis of the corresponding compounds of formula A.

The compound of formula A corresponds to the structure:

where R20is1-C4-alkyl, and-o-o-, R19, -A-a-, -B-b - and R3defined for formula 204.

The compound of formula AA corresponds to the formula A20, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula IA, where-e-E - corresponds to the formula XLIII, XLIV, XLV or XLVII.

Compounds of formula IS, A205D and AE, respectively, correspond to the formula A, AA and AB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula A can be obtained by reacting the corresponding compounds of formula A with alcohol and acid.

The compound of formula A corresponds to the structure:

where R19, R20, -E-e-, -a-a-, -B-B - and R3defined for formula 205.

The compound of formula AA corresponds to the formula A, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula IA, where-e-E - corresponds to the formula XLIII, XLIV, XLV or XLVII.

Compounds of formula IS, A206D and AE, respectively, correspond to the formula A, AA and AB, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula A, can be obtained by interaction of the corresponding compounds of formula A with halide trialkylamine.

The connection forms of the crystals A corresponds to the structure:

where R25is1-C4alkyl and-o-o-, R19, R20, -A-a-, -B-b - and R3defined for formula A.

The compound of formula AA corresponds to the formula A, where R21, R22and R23independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula IA, where-e-E - corresponds to the formula XLIII, XLIV, XLV or XLVII.

Compounds of formula IS, A207D and AE, respectively, correspond to the formula A, AA and AB, where each one-And-A - and-B-B - represents-CH2-CH2and R3represents hydrogen. Compounds covered by the formula A can be obtained by reacting the corresponding compounds of formula A with diallylmalonate.

The compound of formula A208 corresponds to the structure:

where-e-E-, R80and R90defined for formula XLII; -a-a-, -B-b - and R3defined in formula 104; and R19, R20, -A-a-, -B-b - and R3defined for formula 205.

The compound of formula AA corresponds to the formula A208, where R21and R22independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula IA, where-e-E - corresponds to the formula XLIII, XLIV, XLV or XLVII.

The compound of formula AS corresponds to the formula A208, where R80 and R90together with the ring carbon to which they are attached, represent a keto or:

where X, Y1, Y2and(17) defined above.

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

Compounds of formula IE, A208F, A208G, AN and A208J, respectively, correspond to the formula A208, AA, AV, AS and A208D, where each one-And-A - and-B-B - represents-CH2-CH2and R3represents hydrogen. Compounds covered by the formula A208, can be obtained by thermal decomposition of the corresponding compounds of formula A.

The compound of formula A corresponds to the structure:

where R80and R90defined for formula XLI, and-e-E-, -a-a-, -B-b - and R3defined in the formula 205.

The compound of formula AA corresponds to the formula A, where R21and R22independently selected from hydrogen, halogen and lower alkyl.

The compound of formula IV corresponds to the formula IA, where-e-E - corresponds to the formula XLIII, XLIV, XLV or XLVII.

The compound of formula AS corresponds to the formula IV, where-e-E - corresponds to the formula XLIV.

The compound of formula A209D corresponds to the formula A208, where R80and R90together with the ring carbon to which they Preece is United, represent a keto or:

where X, Y1, Y2and(17) defined above.

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

Compounds of formula A209F, A209G, AN, A209J, A209L and AM, respectively, correspond to the formula A, AA, AB, AC, A208D and AE, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula A can be obtained by hydrolysis of the corresponding compounds of formula A208.

The compound of formula a corresponds to the structure;

where R80and R90defined for formula XLI, and Vice-a-a-, -B-b - and R3defined for formula IV.

The compound of formula AA corresponds to the formula a, where R80and R90together with the ring carbon to which they are attached, represent a keto or:

where X, Y1, Y2and(17) defined above.

The compound of formula IV corresponds to the formula AA where the Deputy XXXIV corresponds to the structure XXXIII:

The compound of formula AS corresponds to the formula IA, where R80and R90together form keto.

Compounds of formula A210D, AE, A210F and A210G, according to the respectively, correspond to the formula a, AA, AV and AS, where each one-And-A - and-B-B - represents-CH2-CH2-, a R3represents hydrogen. Compounds covered by the formula 210, can be obtained by epoxidation of compounds of formula 209, in which-e-E - is.

The compound of formula I corresponds to the formula:

where-a-a-, -B-b - and R3defined above.

The compound of formula AA corresponds to the formula A, where R80and R90together form a keto or:

where X, Y1, Y2and(17) defined above.

The compound of formula IV corresponds to the formula IA, in which Deputy XXXIV corresponds to the structure XXXIII:

The compound of formula AS corresponds to the formula IA, where R80and R90together form keto.

Compounds of formula A211D, AE, A211F and A211G, respectively, correspond to the formula A, AA, AV and AS, where each one-And-A - and-B-B - represents-CH2-CH2-, and R3represents hydrogen. Compounds covered by the formula A, can be obtained by oxylane the corresponding compounds of formula a, or by epoxidation of the corresponding compounds of formula A, where-e-E - is. Connection fo the mules A can be converted into compounds of formula I by the way described below.

The compound of formula L corresponds to the structure:

where R11is1-C4alkyl, and-And-And -, - - -, R1, R2, R3, R8and R9defined above.

The compounds of formula LA correspond to the formula L, where R8and R9together with the carbon to which they are attached, represent:

where X, Y1and Y2defined above.

The compounds of formula LB correspond to the formula L, where R8and R9match the structure XXXIII

Compounds of the formula LC, LD, LE correspond to the formula L, LA and LB, respectively, where each of the-a-a - and-B-B - represents-CH2-CH2-, a R3represents hydrogen.

According to the description of the specific reaction schemes below, it will be possible to determine which of these compounds are most suitable for this reaction scheme. Compounds of the present invention can be used as intermediates for obtaining epoxyoctane and other steroids.

Other objectives and features of the present invention in part obvious and in part described below.

Brief description of drawings

Figure 1 presents the diagram of a method of biological transformation canrenone or a derivative thereof is about canrenone in the corresponding 11α -gidroksosoedinenii;

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

figure 3 presents the scheme is particularly preferred method of biological transformation /11-α-hydroxylation canrenone and derivatives canrenone;

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

figure 5 presents the size distribution of particles canrenone, sterilized in the fermenter for the transformation method, illustrated in Figure 3.

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

Description of the preferred embodiments of the present invention

In accordance with the present invention have been developed various new schemes of ways to get epoxyoctane and other compounds corresponding to the formula I:

where:

-A-a - represents the group-CHR4-CHR5or CR4=CR5-;

R3, R4and R5independently selected from the group comprising hydrogen, halogen, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy;

R1PR is dstanley α oriented lower alkoxycarbonyl or hydroxyalkyl radical; and

- - Represents a group-CHR6-CHR7- or alpha - or beta-oriented group:

where R6and R7independently selected from the group comprising hydrogen, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, aryloxyalkyl, cyano, aryloxy; and

R8and R9independently selected from the group comprising hydrogen, hydroxy, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, aryloxyalkyl, cyano, aryloxy; or R8and R9taken together, represent a carbocyclic or heterocyclic ring structure, or R8or R9taken together with R6or R7represent carbocyclic or heterocyclic ring structure fused with Pyh ring D.

If it is not specifically mentioned, organic radicals, referred to in the present description "lower"contain a maximum of 7, preferably from 1 to 4 carbon atoms.

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, isobut is l, sec-butyl and tert-butyl; especially preferred are methoxycarbonyl, etoxycarbonyl and isopropoxycarbonyl. Lower alkoxy radical is preferably a radical derived from one of the above1-C4alkyl radicals, especially from the primary1-C4-alkyl radical; especially preferred is methoxy. Lower alkanoyl radical is preferably a radical derived from direct alkyl having from 1 to 7 carbon atoms; especially preferred are formyl and acetyl.

Methylene bridge in the 15,16-position is preferably β-oriented.

A preferred class of compounds, which can be obtained by the methods of the present invention, are 20-spiroxazine compounds described in U.S. Patent No. 4559332, i.e. compounds corresponding to formula IA

where:

-A-a - represents the group-CH2-CH2- or-CH=CH-;

-B-B - represents the group-CH2-CH2- or alpha - or beta-oriented group of formula IIIA:

R1represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonyl radical;

X represents two hydrogen atoms, oxo or =S;

Y1and Y2in atie together represent the oxygen bridge-O -, or

Y1represents hydroxy, and

Y2represents hydroxy, lower alkoxy or, if X represents H2also lower alkanoyloxy.

Preferably, 20-spiroxazine compounds obtained by the new methods of the present invention, are compounds of formula I, where Y1and Y2taken together represent the oxygen bridge-O-.

Especially preferred compounds of formula I are compounds in which X represents oxo. 20-pyroxenoid compounds of formula 1A, where X represents oxo, most preferred are those compounds in which Y1together with Y2represent the oxygen bridge-O-.

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

The preferred compounds are alkali metal salts, especially potassium salts of compounds of formula IA, where R1is alkoxycarbonyl, X is oxo, and each of the Y1and Y2represents hydroxy.

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

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

9α,11α-epoxy-7α-etoxycarbonyl-20-spirox-4-ene-3,21-dione;

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

and 1,2-degidro-analogues of each of these compounds;

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

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

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

and 1,2-degidro-analogues of each of these compounds;

9α,11α-epoxy-7α-metuximab the Il-17β -hydroxy-3-oxo-pregn-4-ene-21-carboxylic acid;

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

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

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

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

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

and alkali metal salts, especially potassium or ammonium salts of each of these acids, and the corresponding 1,2-degidro-analogues of each of the above-mentioned 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 esters;

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

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

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

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

<> 9α,11α-epoxy-17β-hydroxy-17α(3-hydroxypropyl)-3-oxo-Drost-4-ene-7α-carboxylic acid methyl, ethyl and isopropyl esters;

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-bitmeyen-androst-4-EN-3-one,

including 17α-(3-acetoxypropionyl)and 17α-(3-formyloxy-propyl)-analogues of the above androstenone connections

and 1,2-degidro-analogues of all kinds of the above compounds androst-4-EN-3-one and 20-spirox-4-EN-3-one.

Chemical names of the compounds of formulas I and IA and compounds analogues having the same characteristic structural features are provided in accordance with accepted nomenclature, namely: names for compounds in which Y1together with Y2represents-O come from 20-spirostane (for example, the compound of formula IA, where X represents oxo and Y1taken together with Y2represents-O, is called the "20-spiroctan-21-he"; the name for compounds in which each of the Y1and Y2is hydroxy, and X is oxo, takes place from 17β-hydroxy-17α-pregnen-21-carboxylic acid; nazvanie for connections in which each of the Y1and Y2is hydroxy and X represents two hydrogen atoms, takes place from 17β-hydroxy-17α-(3-hydroxypropyl)-androstane. Because of the circular shape and forms an open circuit, i.e. lactones and 17β-hydroxy-21-carboxylic acids and their salts, respectively, have such a close affinity with each other that the latter may be only the hydrated form first, it should be noted that in the foregoing and subsequent descriptions, if it is not specifically mentioned, these forms, as in the final products of formula I and the source and the intermediate compounds of similar structure, in all cases mentioned together.

In accordance with the present invention was developed several separate circuits for producing compounds of formula I in high yield and at a reasonable cost. Each of these circuits synthesis provides for a series of intermediate compounds. A number of these intermediate compounds are new compounds, and methods of obtaining these intermediate compounds are new ways.

Scheme 1 (using the original canrenone or related compounds)

In one preferred schemes for preparing compounds of the formula I as starting compound used mainly canrenone or Rhodes is venous connection the corresponding formula XIII (or, alternatively, the method comprises using as the source connection of Androstenedione or related compounds)

where:

-A-a - represents the group-CHR4-CHR5or CR4=CR5-;

R3, R4and R5independently selected from the group comprising hydrogen, halogen, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano, aryloxy;

-B-B - represents the group-CHR6-CHR7- or alpha - or beta-oriented group:

where R6and R7independently selected from the group comprising hydrogen, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, aryloxyalkyl, cyano, aryloxy; and

R8and R9independently selected from the group comprising hydrogen, hydroxy, halogen, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, aryloxyalkyl, cyano, aryloxy; or R8and R9taken together are keto, carbocyclic or heterocyclic ring structure, or R8or R9taken together with R6or R7represent carbocyclic or heterocyclic ring structure, to generowania with Pyh ring D.

Using the method of biological transformation type of the method, illustrated in figures 1 and 2, 11-hydroxy-group in α-orientation is introduced into the compound of formula XIII, resulting in a receive connection formula VIII:

where-a-a -, - - -, R3, R8and R9defined in formula XIII.

The compound of formula XIIIA has preferably the structure:

and 11α-hydroxy-product has the structure

in each of them:

-A-a - represents the group-CH2-CH2- or-CH=CH-;

-B-B - represents the group-CH2-CH2or αor β-oriented group:

R3represents hydrogen, lower alkyl or lower alkoxy;

X represents two hydrogen atoms, oxo or =S;

Y1and Y2taken together represent the oxygen bridge-O -, or

Y1represents hydroxy, and

Y2represents hydroxy, lower alkoxy or, if X represents H2also lower alkanoyloxy;

and salts of compounds in which X represents oxo, a Y2represents hydroxy. More preferably, when the compound of the formula VIIIA obtained in this reaction corresponds to formula VIIIA, where each one-And-A-, -B-B - represents-CH2 -CH2-; R3represents hydrogen; Y1and Y2and X are defined in formula XIIIA; and R8and R9taken together, form 20-spiroxamine structure:

.

Preferred microorganisms that can be used in this stage hydroxylation, are Aspergillus ochraceus NRRL 405, Aspergillus ochraceus ATCC 18500, Aspergillus niger ATCC 16888 and ATCC 26693, Aspergillus nidulans ATCC 11267, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillus B. 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 have activity 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 ATSS 13144, Botryosphaeria obtusa IMI 038560, Calonectria 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 ATCC 22921, Curvularia lunata ACTT 12017, Cylindrocarpon radicicola ATCC 1011, Epicoccum humicola ATCC 12722, Gongronella 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 ATCC 19070, Paecliomyces carneus ATCC 46579, Penicillum 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 ACC 25489, Syncephalastrum racemosum ATCC 18192, Thamnostylum piriforme ATCC 8992, Thielavia terricola ATCC 13807 and Verticillium theobromae ATCC 12474.

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), pp. 103 -- 110), Aspergillus ustus (Acta Environ. Pol., Ser.B. (1973), 5(2), pp. 103 -- 110), Fusarium frost (Acta Microbiol.Pol., Ser.B. (1973), 5(2), pp. 103 -- 110), Verticillium glaucum (Acta Microbiol.Pol., Ser.B (1973), 5(2), pp. 103 -- 110) and Rhizopus nigricans (Biochem J.Steroid. (1987), 28(2), 197-201).

11β-Hydroxy-Androstenedione derivatives and maxidone can be obtained by means of biological transformation described in Examples 19A and 19B, respectively. By analogy, the authors of the present invention, it has been assumed that the corresponding β-hydroxy-isomer of compounds of formula VIII with instead of C11-α-hydroxy-Deputy C11-β-hydroxy-Deputy, can be obtained in a similar way to biological transformation using suitable microorganisms capable of 11β-hydroxylation, such as one or more of the microorganisms described in this application.

Before carrying out a large-scale fermentation for hydroxylation carninoma or other substrates of formula XIII, receive an inoculum of cells in the seed si is theme for fermentation, containing the seed fermenter, or two or more series-connected seed fermenters. Working original spore suspension is introduced into the first seed fermenter together with a nutrient solution for cell culture. If it is desirable or necessary that the amount of inoculum exceeded the amount that is produced in the first seed fermenter, the volume of inoculum can be increased in arithmetic or geometric progression by passing through the other consistently located fermenters in seeding unit for fermentation. Preferably, the inoculum produced in the seed system for fermentation, had sufficient and contained viable cells to achieve rapid initiation of the reaction in the production fermenter; and that successive cycles of production were relatively short and to the fermenter had high performance. Regardless of the number of vessels in the seed fermentors, the second and subsequent seed fermenters should be preferably of such size that the degree of dilution in each stage of this series was basically 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 Il is another substrate of formula XIII is loaded into the production fermenter together with the inoculum and nutrient solution and carry out the hydroxylation reaction.

The spore suspension loaded in the sowing system for fermentation, served from a vessel with a working source solution suspensions of spores taken from a variety of vessels, containing already working harvested the source Fund of cells, which, before use, is stored in cryogenic conditions. The working Fund of the source cells, in turn, is derived from the uterine Fund source cells, which is obtained as follows. Sample dispute, obtained from the appropriate source, for example from ATSS first suspended in an aqueous medium, such as, for example, saline, nutrient solution or the solution of surface-active substances (for example, nonionic surfactants such as tween-20, at a concentration of about 0.001% of the mass.) and this suspension was dispensed into cups for cultivation, each of which contains a solid nutrient mixture, usually on the basis digitalimage polysaccharide such as agar, to breed controversy. 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 such as peptone; from about 0.05% to about 0.5% of the mass. source of phosphorus, such as phosphate, ammonium or alkali metal, such as declivitous; from about 0.25 % to about 2.5% of the mass. yeast lysate or extract (or other East is cnica amino acids, such as meat extract or broth with cardio-cerebral extract; from about 1% to about 2% of the mass. agar or other neytralinogo polysaccharide. Additionally, but not necessarily, solid nutrient mixture may contain and/or contains from about 0.1% to about 5% of the mass. extract of malt. 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 an alkali metal hydroxide or phosphoric acid. Suitable solid media for cultivation are the following environments:

1. Solid medium #11% glucose, 0.25% of yeast extract, 0.3% of K2HPO4and 2% agar (Bacto); pH was brought to 6.5 with 20% NaOH
2. Solid medium #22% peptone (Bacto), 1% yeast extract (Bacto), 2% glucose and 2% agar (Bacto); the pH is brought to 5 with 10% H3PO4
3. Solid medium #30.1% peptone (Bacto), 2% malt extract (Bacto), 2% glucose and 2% agar (Bacto); pH=5,3
4. Liquid medium5% black molasses, 0.5% of the liquid corn extract, 0.25% of glucose, and 0.25% NaCl and 0.5% KN2PO4pH=5,8
5. Mycological agarDifco (low pH)

The number of agar cups used for receiving the Oia's main source of cellular Fund, can be selected depending on the future requirements for the source uterine material, but generally, this number is from about 15 to about 30 thus prepared cups. After the cultivation period, for example within 7-10 days, cups otskrebaya to collect spores in the presence of aqueous media, saline solution or buffer, and the resulting stock of the original suspension was dispensed into small vessels, for example, 1 ml of the suspension was placed in each of the sets of 1.5 ml of blood vessels. To get a working initial suspension of spores for research or for industrial fermentation of the contents of one or more of these vessels with uterine source material of the second generation can be distributed cups with agar and incubated in a manner analogous to the method described above to obtain uterine initial suspension of spores. If there are traditional industrial production, to obtain a working source material of the second generation can be used at least from 100 to 400 cups. The contents of each Cup otskrebaya in a separate vessel with a working source material, where each vessel typically contains 1 ml produced inoculum. For permanent preservation as uterine original suspension, and inoculate the second generation store is preimushestvenno in the vapor phase in the vessel for cryogenic storage containing liquid N2or other cryogenic liquid.

In the method, illustrated in figure 1, get water medium for cultivation, which contains a nitrogen source such as peptone, yeast derivative or its equivalent, glucose, and a source of phosphorus, such as phosphate salt. Spores of the microorganism cultivated in this environment in the seed fermentation system. The preferred microorganism is Aspergillus ochraceus NRRL 405 (ATCC 18500). Then thus obtained sowing medium is introduced into the fermenter for producing together with the substrate of formula XIII. The broth for fermentation and stir aeronaut during the time interval sufficient to complete reaction to the desired degree of completion.

Environment for seed fermenter, preferably consists of an aqueous mixture which contains between about 0.5% and about 5% of the mass. glucose, from about 0.05% to about 5% of the mass. a nitrogen source such as peptone; from about 0.05% to about 0.5% of the mass. source of phosphorus, such as phosphate, ammonium or alkali metal, such as the monobasic ammonium phosphate or dicale-phosphate; from about 0.25% to about 2.5% of the mass. yeast lysate or extract (or other source of amino acids, such as stillage extract ; from about 1% to about 2% of the mass. agar or other neytralinogo polysaccharide. Especially preferred environment for the cult of the growing 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. autorisierung 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. source of phosphorus, such as monobasic ammonium phosphate. Particularly economical methods were developed using another preferred seed culture, 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. autorisierung 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 particularly economical source of protein, peptides, carbohydrates, organic acids, vitamins, metal ions, trace elements and phosphate. Instead of liquid corn extract, or in addition to it can be used a solution of pulp from other grains. the pH of this medium is preferably adjusted to values of from about 5.0 to about 7.0 and, for example, by adding an alkali metal hydroxide or phosphoric acid. If as a source of nitrogen and carbon used liquid corn extract, the pH is preferably adjusted to values of from about 6.2 to about 6.8. The medium containing peptone and glucose, preferably adjusted to a pH of from about 5.4 to about 6.2. Environments for Kul is iferouane, suitable for use in the seed fermentation are:

1. Environment #12% peptone, 2% autorizovanych yeast (or yeast extract and 2% glucose; pH increased to 5.8 using 20% NaOH
2. Environment #23% liquid corn extract, 1.5% of yeast extract, 0.3% of monobasic ammonium phosphate and 3% glucose, pH is brought to 6.5 with 20% NaOH.

Spores of the microorganism is introduced into the environment from a vessel, usually containing approximately 109spores per ml of suspension. The optimal productivity of seed generation is achieved in the case when the dilution of the culture medium at the beginning of the cultivation of the seed culture, the population density of the dispute is not reduced below about 107on ml. Preferably, the spores are cultivated in the seed fermentation system as long as the volume of precipitated mycelium (PMV) in the seed fermenter will not be at least about 20%, and preferably from about 35% to about 45%. Since the loop in the vessel for fermentation inoculum (or in any vessel of the many vessels that constitute a system for seed fermentation) depends on the initial concentration in the vessel, it may be preferable to perform the two-or three-stage seeding the second fermentation to speed up the whole process. However, it is desirable to avoid the use of much more than three fermenter in the system, as in the case when the fermentation inoculum includes an excessive number of stages, it may adversely affect the activity of the process. Fermentation of the seed culture is conducted under stirring at a temperature ranging from about 23 to about 37°and preferably in the range from about 24 to about 28°C.

The culture of the seeding system fermentation is injected into the production fermentor with working environment for cultivation. In one of the embodiments of the invention as a substrate for the reaction is non-sterile canrenone or other substrate of formula XIII. Preferably, if the substrate is added in the production fermenter in the form of 10%-30% of the mass. suspension in the medium for cultivation. To increase the surface area available for reaction 11α-hydroxylation, before the introduction of the substrate of formula XIII in the fermenter particle size of the substrate is reduced by passing it through an offline micronizer (mill for fine grinding). Also separately injected sterile source nutrient solution containing glucose, and a second sterile nutrient solution containing yeast derivative, such as avtorizovanniy yeast (or the equivalent and inoculate composition, based on alternative sources, such as the extract of the bards). This environment consists of 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 such as peptone; from about 0.05% to about 0.5% of the mass. source of phosphorus, such as phosphate, ammonium, or an alkali metal, such as declivitous; from about 0.25% to about 2.5% of the mass. yeast lysate or extract (or other source of amino acids, such as stillage extract; from about 1% to about 2% of the mass. agar or other neytralinogo polysaccharide. Especially preferred working environment for culturing 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. autorisierung 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. 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. autorisierung 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 the production of fermentation is preferably adjusted in the manner described above for the environment, prednaznachen the th for seed fermentation, the most preferred are the limits of pH values specified for the medium containing peptone/glucose, and for environment containing liquid corn extract, respectively. Media for culturing suitable for reactions of biological transformation are listed below:

1. Environment #12% peptone, 2% autorizovanych yeast (or yeast extract and 2% glucose; pH increased to 5.8 using 20% NaOH
2. Environment #21% peptone, 1% autorizovanych yeast (or yeast extract and 2% glucose; pH increased to 5.8 using 20% NaOH
3. Environment #30.5% peptone, 0.5% of autorizovanych yeast (or yeast extract) and 0.5% glucose, pH increased to 5.8 using 20% NaOH.
4. Wednesday #43% liquid corn extract, 1.5% of yeast extract, 0.3% of monobasic ammonium phosphate and 3% glucose, pH is brought to 6.5 with 20% NaOH.
5. Wednesday #52,55% liquid corn extract, 1,275% yeast extract, 0,255% monobasic ammonium phosphate and 3% glucose, pH is brought to 6.5 with 20% NaOH.
6. Wednesday #62.1% of the liquid corn extract, 1,05% yeast extract, 0,21% monobasic ammonium phosphate and 3% glucose, pH is brought to 6.5 with 20% NaOH.
/p>

Non-sterile solution canrenone and sterile nutrient solution download by supply chain in the production fermenter from about five to about twenty, preferably from about ten to about fifteen portions, and preferably generally equal portions of each ingredient throughout the production cycle. However, before inoculation broth for seed fermentation, the substrate is first injected, preferably in a quantity sufficient to achieve a concentration from about 0.1 wt%. up to about 3 wt. -%, and preferably from about 0.5% wt. to about 2 wt. -%, and then periodically add, basically every 8-24 hours, to achieve a cumulative amount from about 1% to about 8% of the mass. If the additional amount of substrate added every 8 hours, then the total will add a little less, for example from 0.25%to 2.5 wt. -%, than in the case when the substrate is added only through the day. In the latter case, it may be necessary cumulative adding canrenone ranging from 2% to about 8% of the mass. Additional nutrient mixture, is added during the fermentation reaction is preferably 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. sterile yeast extract or other sterile source drug is avago derived (or other amino acid source). As the substrate is fed into the production fermentor, shown in figure 1, is non-sterile, to suppress the growth of unwanted microorganisms in the fermentation broth for periodically adding antibiotics. Antibiotics such as kanamycin, tetracycline and cephalexin, can be added without any adverse effects on growth and biological transformation of microorganisms. These antibiotics are injected into the broth for fermentation, preferably in a concentration, for example from about to 0.0004% to about 0.002 per cent on the total mass of the broth containing, for example, from about is 0.0002% to about 0,0006% kanamycin sulfate, from about is 0.0002% to about 0,006% tetracycline·HCl and/or from about 0,001% to about 0.003 per cent cephalexin on the total mass of the broth.

Usually the production fermentation lasts about 80-160 hours. Thus, portions of each of the substrates of the formula XIII and nutrient solutions usually add approximately every 2 to 10 hours, and preferably after every 4-6 hours. In the system of seeding the fermentation in the production fermenter is preferably also enter the antifoam.

In the method, illustrated in figure 1, the inoculum load in the production fermenter, preferably in an amount of from about 0.5% to about 7%, more preferably from about 1% to about 2% by volume full of the mixture in the farm is Tere, as the concentration of glucose support 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 are preferably carried out portions from about 0.05% to about 0.25% by total weight of the load. Fermentation temperature is preferably regulated in the range from 20°to 37°C, preferably from 24°up to about 28°With, however, it may be desirable to gradually reduce the temperature in the reaction, for example, on 2°but at the same time to support the amount of precipitated mycelium (PMV) below about 60%, and more preferably below about 50%, which helps to prevent increase in the viscosity of the broth during fermentation, which may prevent adequate mixing. If the growth of biomass beyond the surface of the liquid, the substrate is present in the biomass, may be outside the reaction zone and to be available for hydroxylation reactions. To maintain productivity, it is desirable that PMV was reached 30-50%, preferably 35-45% in the first 24 hours of fermentation reaction, and then the conditions are preferably adjusted to control subsequent growth within the limits established above. During the reaction the pH of the medium for fermentation adjusted in the range from about the olo 5.0 to about 6.5, preferably from about 5.2 to about 5.8 and the contents of the fermenter was stirred at a speed ranging from about 400 to about 800 rpm, the Level of dissolved oxygen of at least about 10% of saturation is achieved by aeration of the party at about 0.2 to 1.0 rpm rpm, the pressure in the head part of the fermenter is approximately in the range from atmospheric pressure to about 1.0 bar, and more preferably of approximately 0.7 bar. The speed of mixing can also be increased if necessary to maintain minimum levels of dissolved oxygen. Dissolved oxygen support, mainly at a level considerably greater than about 10%, and in fact up to about 50% in order to stimulate the transformation of the substrate. Maintaining the pH in the range 5.5±0.2 is also optimal condition for biological transformation. Foaming regulate, if necessary, by adding commonly used antifoam. After adding the entire substrate reaction is preferably continued until until the molar ratio of the product of formula VIII to the remaining unreacted substrate of formula XIII will not be at least about 9 to 1. This transformation can be achieved in the process 80-160-hour cycle described above.

It was found that the high degree of transformation involves the depletion levels source of nutrients below the initial level load, therefore, by regulating the speed of aeration and the rate of mixing is possible to prevent splashing of the substrate from the liquid broth. In the process, illustrated in figure 1, the level of nutrients is exhausted, and then is maintained at the level not exceeding about 60%, preferably about 50%, from the level of the initial load, and in the process, illustrated in figure 2 and 3, the level of nutrients is reduced to the level and processor cores are supported at a level not exceeding about 80%, preferably about 70% of the initial load. The rate of aeration, preferably does not exceed about 1/rpm, and more preferably is in the order of about 0.5 rpm rpm; and the speed of stirring is preferably not more than 600 rpm

A particularly preferred method for obtaining compounds of formula VIII are illustrated in figure 2. The preferred microorganism for 11α-gidroksilirovanii the compounds of formula XIII (for example, canrenone) is Aspergillus ochraceus NRRL 405 (ATCC 18500). In this way, the medium for culturing 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, it can also be used and other production environments DL the cultivation. Sowing culture comes primarily by way illustrated in Fig. 1, using any medium for seed fermentation described in this application. The suspension is not crushed canrenone or other substrate of formula XIII in the medium for culturing get in aseptic conditions in the mixer, preferably in a relatively high concentration factor of about 10% to about 30% by weight of the substrate. Preferably, the aseptic condition of receiving may include sterilization or pasteurization of the suspension after mixing. The full amount of a sterile suspension of substrate required to obtain a parcel of product injected into the production fermenter at the beginning of the cycle or by periodic supply chain. The particle size of the substrate is reduced by wet grinding in a pump with a shear action, working in online mode, which delivers the slurry to the production fermenter, which avoids the need to use an offline micronizer. If aseptic conditions provided by pasteurization, not sterilization, the degree of agglomeration may be small, but the use of a pump with a shear action may be desirable for positive regulation of the particle size. Sterile environment for cultiva the Finance and glucose solution is injected into the production fermentor, basically, in a way similar to that described above. All nutrient components prior to their introduction into the production fermentor is sterilized, and therefore the use of antibiotics is not required.

In the preferred implementation of the method, illustrated in figure 2, the inoculum is injected into the production fermenter in an amount of from about 0.5% to about 7%, the temperature of the fermentation is from about 20°to 37°C, preferably from about 24°up to about 28°and the pH 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 figure 1, the temperature is preferably adjusted to regulate the growth of the biomass so that the PMV does not exceed 55-60%. Initial load of glucose, preferably, is from about 1% to about 4 wt. -%, and more preferably of 2.5%is 3.5 wt. -%, however, in the fermentation process, it can be preferably below about 1.0% of the mass. An additional amount of glucose is fed periodically from about 0.2% to about 1.0% of full mass of the charging party so that the concentration of glucose in the fermentation was maintained in the range from about 0.1% to about 1.5 wt. -%, and PR is doctitle from about 0.25% to about 0.5% of the mass. Sources of nitrogen and phosphorus can, but need not, be submitted together with glucose. However, because downloading just canrenone is made at the beginning of the cycle for the Patria, it is necessary to supply the nitrogen - and phosphorus-containing nutrients can also be carried out simultaneously, which can be used to add to the reaction only glucose. The speed and type of mixing can vary substantially. Moderate stirring stimulates mass exchange between the solid substrate and an aqueous phase. However, to prevent degradation of myelin microorganisms need to use the mixer with a small shear effort. The optimum speed of mixing varies from 200 to 800 rpm depending on the viscosity of the culture broth, the oxygen concentration and the conditions of mixing, which is influenced by the configuration of the vessel, baffles and agitators. Usually the preferred speed of mixing is in the range from 350 to 600 rpm, Preferably, the blades for mixing realize the function of axial pumping downwards, which provides good mixing fermentation of biomass. This party preferably aeronaut when the speed component of from about 0.3 to about 1.0 V/V/min, and preferably from about 0.4 to 0.8 to about rpm rpm and the pressure in the head part of EN zymes the EPA is preferably, from about 0.5 to about 1.0 bar on the scale of reference. The temperature of mixing, aeration and the pressure is preferably regulated to maintain the dissolved oxygen level of at least about 10% by volume in the process of biological transformation. The length of the entire cycle for this party is usually from about 100 to about 140 hours.

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

In the preferred method, illustrated in Figure 3, full download party and nutrient solution is sterilized in a manufacturing apparatus for fermentation, and then introducing the inoculum. Nutrient solution which can be used, and preferred of these solutions are basically the same as in the method illustrated in figure 2. In this embodiment of the invention the shear force of the blades of the agitator breaks agglomerates of the substrate, in one way or another are formed after sterilization. It was found that this reaction proceeds favorably, if the particle size canrenone is less than about 300 microns and at least 75% of the mass. all of the particles have a size less than 240 microns. It was found that the use of a suitable mixer, such as a disk turbine agitators, with adequate speed of the order of 200-800 rpm, with a maximum speed component of at least about 400 cm/sec, provides a shear rate sufficient to maintain the specified values of particle size, despite the agglomeration of particles, which usually occurs after sterilization in the production fermenter. Other operations of the method illustrated in Figure 3, are basically the same as in the method illustrated in figure 2. The methods are illustrated on Figure 2 and 3, have some significant advantages in comparison with the method illustrated in figure 1. The main advantage is the possibility of using low-cost pit is positive fundamentals, such as liquid corn extract. Other advantages are realized by eliminating the need to add antibiotics, which simplifies the procedure and allows sterilization of the party canrenone or other substrate of formula XIII. Another significant advantage is the ability to use a simple glucose solution instead of complex nutrient solution to be added during the reaction cycle.

In the methods illustrated in figures 1 to 3, the product of formula VIII is a crystalline solid, which, together with the biomass can be separated from the reaction broth by filtration or low speed centrifugation. Alternatively, this product can be extracted from a reaction broth using organic solvents. The product of formula VIII allocate by solvent extraction. For maximum isolation liquid filtrate and a filter with biomass or sludge in the centrifuge process extracting solvent, but usually ≥95% of the product associated with the biomass. Usually, the extraction may be used a hydrocarbon, ester, chlorinated hydrocarbon and ketone solvents. The preferred solvent is ethyl acetate. Other, mainly suitable rastvorityelya toluene and methyl isobutyl ketone. For extraction of the liquid phase may be preferred to use an amount of solvent, approximately equal to the volume of the reaction solution with which it contacts. For product recovery from biomass the biomass suspended in a solvent, preferably in large excess relative to the initial loading of the substrate, for example, 50-100 ml of solvent per gram of initial load canrenone, and the resulting suspension is preferably heated under reflux over a period of time from about 20 minutes to several hours to ensure transfer of the product phase solvent from the cavities 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 water washing and the washing solvent are combined and left for phase separation. The product of formula VIII allocate by crystallization from solution. To maximize output mycelium twice subjected to contact with fresh solvent. After deposition, the product is separated from the phase of the solvent for a complete separation of the aqueous phase. More preferably the solvent is evaporated under vacuum until then, until the start of crystallization, and then the concentrated extract is cooled to a temperature which s from about 0° With up to about 20°and preferably from about 10°With up to about 15°With, within a period of time sufficient for the deposition and growth of crystals, usually within from about 8 to about 12 hours.

Most preferred are methods, illustrated in figure 2, and particularly the manner illustrated in Figure 3. These methods are carried out at low viscosity and they are suitable for precise control of process parameters such as pH, temperature and dissolved oxygen. In addition, the conditions of sterility easily maintained without the introduction of antibiotics.

The processes of biological transformation is exothermic, which requires heat dissipation using a fermentor with jacket or cooling coil in the production fermenter. Alternatively, the reaction broth can be returned to again cycle through an external heat exchanger. Dissolved oxygen is preferably supported at the level of at least about 5%, and preferably at least about 10% by volume, which is sufficient to provide energy for this reaction and guarantees the transformation of glucose in CO2and H2About by regulating the flow rate of air introduced into the reactor after measuring the level of oxygen in the broth. the pH is preferably maintained within the range of from about 4.5 d is about 6.5.

In each of the alternative ways 11-hydroxylation of the substrate of formula XIII productivity of this method is limited by the mass transfer from the solid substrate in the aqueous phase or to the phase boundary, where it is obvious the reaction occurs. As mentioned above, productivity is largely limited by the rate of mass transfer, provided that the average particle size of the substrate is reduced to less than about 300 microns, and at least 75% of the particles have a size less than 240 microns. However, the productivity of these processes may be further enhanced in some alternative embodiments of the invention, which provides the main load canrenone or other substrate of formula XIII in the production fermenter in an organic solvent. In accordance with this variant, the substrate is dissolved in is not miscible with water solvent and mixed with the original aqueous culture medium for the preservation and surface-active substance. Suitable water-immiscible solvents are, for example, DMF, DMSO, C6-C12fatty acid, With6-C12-n-alkanes, oils, sorbitan and aqueous solutions of surfactants. Stirring this download helps producing the emulsion reaction system having an extended area of Espanol surface for mass transfer of the substrate from the organic liquid phase in the reaction zone.

In the second embodiment of the invention, first dissolve the substrate in mixing with the water solvent, such as acetone, methyl ethyl ketone, methanol, ethanol or glycerol, at a concentration, mainly in excess of its solubility in water. Upon receipt of the original substrate solution at elevated temperature, the solubility increases, which increases the amount of soluble forms of the substrate introduced into the reactor, and thereby increases the payload of the reactor. Warm substrate solution is loaded into a production reactor for fermentation with relatively cold water boot containing medium for cultivation and inoculate. When mixing the substrate solution with the aqueous medium is deposited substrate. However, in conditions of significant swarnakumari and moderately intensive mixing instead of crystal growth is the predominant formation of centers of crystallization, the formation of very small particles with a large surface area. Large surface area promotes mass transfer between the liquid phase and the solid substrate. In addition, the equilibrium concentration of substrate in the aqueous liquid phase also increases in the presence of mixing with the water solvent. In line with this increased productivity.

Although Dunn is the first microorganism does not necessarily have to be tolerant to high concentration of organic solvent in the aqueous phase, however, it is desirable to use a concentration of ethanol, for example, ranging from about 3% to about 5% of the mass.

The third embodiment of the invention is the solubilization of the substrate in an aqueous solution of cyclodextrin. Typical cyclodextrin is 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 mixture substrate: cyclodextrin may be added, in terms of reimbursement, in the reactor for the biological transformation.

11α-Hydroxybenzene and other reaction products 11α-hydroxylation (Formula VIII and VIIIA) are new compounds that can be isolated by filtering the reaction medium and extraction of the product from biomass collected in the medium after filtering. For extraction can be used in standard organic solvents such as ethyl acetate, acetone, toluene, chlorinated hydrocarbons and methyl isobutyl ketone. Then the product of formula VIII can be recrystallized from an organic solvent of the same type. The compounds of formula VIII are used primarily as intermediates for producing compounds of formula I and, primarily, the compounds of formula IA.

Preferably the compounds of formula VIII correspond to the formula VIIIA, where-a-a - and-B-To - represent-CH2-CH2-, R3represents hydrogen, lower alkyl or lower alkoxy, and R8and R9together constitute the 20-spiroxamine ring:

Then, in accordance with the method shown in Scheme 1, the compound of formula VIII is subjected to reaction with a source of cyanide ion in alkaline conditions with obtaining raminosoa the compounds of formula VII

where-a-a -, - - -, R3, R8and R9defined above.

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

where-a-a -, - - -, R3, Y1, Y2and X are defined in formula XIIIA.

R3is preferably hydrogen.

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

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

In the method using cyanohydrin ketone the reaction is carried out in solution, preferably with COI is whether the aprotic polar solvent, such as dimethylformamide or dimethylsulfoxide. The formation of enamine requires the use of at least two moles of a source of cyanide ion per one mol of substrate, and preferably a small excess source of cyanide. The base is preferably a nitrogenous base, such as dialkylamino, trialkylamine, alkanolamine, pyridine or the like, However, can also be used inorganic bases such as carbonates of alkali metals or hydroxides of alkali metals. Preferably, the substrate of formula VIII are initially present in a ratio of from about 20 to about 50 wt. -%, 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 higher temperatures the yield increases. For example, if the base use triethylamine, the reaction is mainly carried out within a temperature range from about 80°to 90°C. At this temperature the reaction proceeds to its completion for a period of from about 5 to about 20 hours. If the base use diisopropylethylamine, the reaction is carried out at 105°and this reaction is finished after 8 hours. Upon completion of the reaction the solvent is removed in vacuum and the residual oil is dissolved in water and neutralized to pH 7 by adding the value of the diluted acid, preferably hydrochloric acid. The product precipitates from this solution, after which it is washed with distilled water and dried with air. The allocation of HCN may be terminated by purging with an inert gas and redeemed in alkaline solution. The dried precipitate is dissolved in chloroform or other suitable solvent, and then extracted with a concentrated acid such as 6N model HC1. The extract was neutralized to pH 7 by adding inorganic bases, preferably the alkali metal hydroxide, and cooled to a temperature in the range of 0°C. the precipitate is washed and dried, and then recrystallized from a suitable solvent, for example acetone, to obtain the product of formula VII, which can be used in the subsequent stage.

Alternatively, the reaction can be carried out in an aqueous solvent system containing mixed with water, an organic solvent, such as methanol, or in a two-phase system containing water and an organic solvent, such as ethyl acetate. In this alternative, the product can be isolated by diluting the reaction solution with water followed by extraction of the product with the use of an organic solvent, such as methylene chloride or chloroform, and then back-extraction of the organic extract using the receiving concentrated mineral acid, for example 2n HCl. Cm. U.S. patent No. 3200113.

In accordance with another alternative method, the reaction can be carried out in mixing with water, a solvent such as dimethylformamide, dimethylacetamide, N-methyl pyrrolidone or dimethyl sulfoxide, and the solution of the reaction product is diluted with water and make it alkaline, for example by adding carbonate of an alkali metal, and then cooled to 0-10°s, which leads to precipitation of the product. Preferably the system is quenched by hypohalite alkali metal or another reagent that is effective to prevent release of cyanide. After filtration and washing with water, the precipitated product can be used in subsequent stages of this process.

In accordance with another alternative method, adaminaby product of formula VII can be obtained by reaction of the substrate of formula VIII in the presence of a source of protons, with an excess of alkali metal cyanide, preferably NaCN, in an aqueous solvent containing an aprotic mixed with water, a polar solvent, such as dimethylformamide or dimethylacetamide. Source of protons is preferably a mineral acid or C1-C5-carboxylic acid, and most preferred is sulfuric acid. It is interesting to note that if zenderoudi reagent is commercially the ski available reagent LiCN in DMF, the discrete source of protons is not necessary to add.

The cyanide ion source, such as a salt of an alkali metal, is loaded into the reactor, preferably in a ratio of from about 2,05 up to about 5 molar equivalents per equivalent of substrate. It is obvious that the mineral acid, or other source of protons, stimulates the accession HCN 4,5 - and 6,7-double bonds and preferably is present in a ratio of at least one molar equivalent of one molar equivalent of the substrate; however, the reaction system must remain alkaline by maintaining the excess amount 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°usually at 60°S-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. Upon completion of the reaction, the reaction mixture is cooled, preferably to about room temperature; and adaminaby the product is precipitated by acidification of the reaction mixture and mixing it with cold water, preferably at a temperature close to the temperature of the ice bath. Acidification, obviously, contributes to the closure of 17-lactone, which tends to break the alkaline conditions prevailing in the reaction of zenderoudi. actionnow the mixture typically is acidified with the same acid, which is present in the reaction and which is preferably sulfuric acid. Water is added preferably in a ratio of from about 10 to about 50 molar equivalents per one mole of the product.

The compounds of formula VII are novel compounds and are used primarily as intermediates for producing compounds of the formula I, especially of formulae IA. Preferably, the compounds of formula VII corresponds to the formula VIIA where-a-a - and-B-To - represent-CH2-CH2-, R3represents hydrogen, lower alkyl or lower alkoxy, and R8and R9together constitute the 20-spiroxamine ring:

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

When the reaction conversion of the compounds of formula VIII in the enamine of Formula VII in the crude product through chromatography watched 7-cyano-derivative compounds of formula VIII. It was suggested that this process of transformation, 7-cyano-soy-Association is an intermediate connection. In addition, it was assumed that the reaction of 7-cyano-derivative leads to the formation of a second intermediate compound, 5,7-dicia the o-derivative compounds of formula VIII, which, in turn, reacts with the formation of complex enefer. Cm. for example, the work R.Christiansen et al., The Reaction of Steroidal 4,6-Dien-3-Ones With Cyanide, Steroids, Vol.1, June 1963, which is introduced in the present invention by reference. These new compounds are also used as chromatographic markers, and are synthetic intermediate compounds. In the preferred embodiment, this phase total synthesis shown in Scheme 1, these intermediate compounds are 7α-cyano-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-21-dicarboxylic acid, γ-lactone and 5β,7α-dicyano-11α,17-dihydroxy-3-oxo-17α-pregnan-21-dicarboxylic acid, γ-lactone.

In the next stage of the synthesis shown in Scheme 1, the enamine of formula VII hydrolyzing with getting diketonato the compounds of formula VI:

where: -a-a -, - - -, R3, R8and R9defined in formula XIII.

For hydrolysis may be used any organic or aqueous mineral acid. Preferred is hydrochloric acid. To increase product yield as a co-solvent preferably used is mixed with water, an organic solvent, such as dimethylacetamide or lower alkanol. The preferred solvent is DIMET acetamid. The acid should be present in a ratio of at least one equivalent per equivalent of substrate of formula VII. In the water system of the substrate enamine VII can be basically turned into a diketone of formula VI for about 5 hours at about 80°C. Conducting the reaction at elevated temperature increases the yield, but the temperature does not play a decisive role. A suitable temperature is chosen based on the volatility of the system of solvents and acids.

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

and diketonaty product corresponds to formula VIA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA. Preferably, R3represents hydrogen.

Upon completion of the reaction the solution is cooled to a temperature of from about 0°to 25°for crystallization of the product. The product in the form of crystals can be recrystallized from a suitable solvent, such as isopropanol or methanol, to obtain the product of formula VI, which can be used in subsequent stages of this method, but usually recrystallization is not necessary stage. The products of formula VI are novel compounds which are mainly used as an intermediate link is to produce compounds of formula I, and especially compounds of the formula IA. Preferably, the compounds of formula VI correspond to the compounds of formula VIA where-a-a - and-B-To - represent-CH2-CH2-, R3represents hydrogen, lower alkyl or lower alkoxy, and R8and R9together constitute the 20-spiroxamine ring:

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

In the most preferred embodiment of the present invention, adaminaby product of the formula VII are obtained from the compounds of formula VIII as described above, and transformed in situ into the diketone of formula VI. In this embodiment of the present invention, the substrate of formula VIII is subjected to reaction with an excess of alkali metal cyanide in an aqueous solvent containing a source of protons, or, optionally, with an excess of cyanohydrin ketone in the presence of a base and LiCI, as described above. However, instead of cooling the reaction mixture, conduct acidification and add water in the ratio required for the deposition of the enamine, while it is preferable to avoid significant cooling of the reaction mixture. Instead, upon completion of the reaction zenderoudi, the mixture is added water and acid, preferably a mineral acid such as sulfuric acid. This amount of added acid is sufficient to neutralize the excess alkali metal cyanide, which usually requires the introduction of at least one molar equivalent of acid per one mole of substrate of formula VIII, and preferably from about 2 to about 5 molar equivalents of the acid to one equivalent of the substrate. However, to avoid significant precipitation reaction is carried out at a sufficiently high temperature and sufficiently high dilution, and the reaction of hydrolysis of the enamine in the diketone is carried out in the liquid phase. Thus, this reaction proceeds with minimal breaks and gives a high yield of product. The hydrolysis is preferably carried out at a temperature of at least 80°S, more preferably in the range from about 90°C to about 100°during the period of time is 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°With up to about 15°C, mostly in an ice bath at a temperature of from about 5°With up to about 10°With, for deposition diketonato product of formula VI. This solid product may be isolated by filtering and reducing the number of impurities is achieved by promilk is water.

In the next stage of the synthesis Scheme 1 diketonate compound of formula VI is subjected to reaction with a metal alkoxide to open ketone bridge between the 4 - and 7-positions by breaking the link between the carbonyl group and the 4-carbon of education α-oriented alkoxycarbonyl substituent in the 7-position and the removal of cyanide from 5-carbon. The product of this reaction is a complex hydroxyamine compound corresponding to the formula V:

where-a-a -, - - -, R3, R8and R9defined in formula XIII, a R1represents the lowest alkoxycarbonyl or hydroxycarbonyl.

The metal alkoxide used in the reaction, corresponds to the formula R10OM, where M represents an alkaline metal, a R10O - corresponds to the alkoxy-substituent R1. The outputs of this reaction are the most satisfactory in the case when the metal alkoxide is methoxide or potassium methoxide, sodium, but can be used and other lower alkoxides. Most preferred is potassium alkoxide. Can also be used phenoxide, other aryloxides and arylsulfonyl. The reaction is usually carried out in the presence of the alcohol corresponding to the formula R10OH, where R10defined above. Can be used and other suitable dissolve the I. The substrate of formula VI is present preferably in an amount of from about 2% to about 12 wt. -%, and more preferably at least about 6% of the mass. R10OM is present preferably in a ratio of from about 0.5 to about 4 mol per one mol of substrate, more preferably from about 1 to about 2 mol per one mol of the substrate, and even more preferably about 1.6 mol per one mol of the 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. Usually the reaction is carried out at the reflux depending on the solvent used.

The time required to achieve equilibrium of the reaction depends on the number of alkoxide, which is added to the reaction mixture, and the method of adding it. The alkoxide may be added in one portion or in several portions, or it can be added continuously. If the alkoxide added in several portions, preferably to about 1.6 equivalents of potassium methoxide were added in two stages. This two-stage adding to the first reaction mixture was added 1 equivalent of potassium methoxide, and then, after 90 minutes, add another 0.6 equivalent of potassium methoxide. This procedure dochstader the nogo add reduces the time to reach equilibrium compared to the addition of 1.6 equivalents of sodium methoxide in one piece.

Since the equilibrium is more favorable conditions for the production of complex hydroxyether at low concentrations diketone, the reaction is preferably carried out at relatively high dilution, for example up to 40:1 for the reaction using sodium methoxide. It was found that significantly higher yields can be obtained when using methoxide potassium instead of sodium methoxide as to minimize the degree reverse zenderoudi, where the reagent is potassium methoxide, is largely sufficient dilution in the range of about 20:1.

In accordance with the present invention, in addition, it was found that the reaction of reverse zenderoudi can be ingibirovany by applying appropriate chemical or physical methods to remove 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 alkoxide of the alkali metal can be carried out in the presence of a precipitating agent for cyanide ion, such as, for example, a salt containing a cation which forms an insoluble cyanide compound. Such salts can be, for example, zinc iodide, ferrous sulfate(3) or, basically, any halide, sulfate or other salt Molochnoe the nutrient or transition metal, which are more soluble than the corresponding cyanide. If iodide zinc is present in a ratio of approximately one equivalent per one equivalent of the substrate diketone, it was found that the productivity of the reaction is considerably increased compared to the reaction carried out in the absence of a halide of an alkali metal.

Even if removal of the ion cyanide use the precipitating agent, it remains preferable to conduct the reaction at a sufficiently high degree of dilution, however, when using this precipitating agent molar ratio solvent:diketonaty the substrate can be significantly reduced compared to reactions where the agent is absent. The selection of complex hydroxyether of formula V can be carried out by methods, either involving or not involving the extraction, as described below.

To stimulate the production of complex hydroxyether of the formula V can also be controlled by the equilibrium of this reaction by removing this complex hydroxyether from the reaction mixture after its synthesis. Remove complex hydroxyether can be carried out Paladino or continuously in such a way as filtering. Remove complex hydroxyether can be used to regulate the equilibrium of the reaction, either alone or in Combi the purpose of chemical or physical removal of cyanide from the reaction mixture. Subsequent heating of the resulting filtrate leads to a shift in the equilibrium of the reaction system in the direction of turning the rest of the diketone of formula VI in a complex hydroxyether formula V.

When the conversion of the diketone of formula VI in a complex hydroxyether of formula V in the raw product was observed in the presence of 5-cyanohydrine in small quantities, components, usually less than about 5% of the mass. It has been suggested that 5-cyanogenesis is an equilibrium intermediate connection between the diketone of formula VI and complex hydroxyether formula V. in Addition, it has been suggested that this equilibrium intermediate compound is formed from the diketone as a result of exposure of methoxide 5.7-oxo-group and protonation of enolate and complex hydroxyether by attaching ion by-product of cyanide to the functional 3-keto-Δ4,5-the group of complex hydroxy-ether by the reaction of Michael.

In addition, the crude product through chromatography was observed 5-cyano-7-acid and 17-alkoxide complex hydroxyether formula V. it has Been suggested that 5-cyano-hydroxyamino intermediate compound reacts with the ion by-product of cyanide (present in the reaction delanerolle, which introduces double Δ4,5rain is z) production of 5-cyano-7-acid. It has been suggested that exposure to cyanide ion leads to dialkylamino 7-ester group 5-cyano-hydroxyether with the formation of 5-cyano-7-acid and the corresponding Alternaria.

In addition, it has been suggested that an unstable intermediate compound 17-alkoxide, is formed as a result of the impact of methoxide on 17-spirolactone complex hydroxyether (or preceding intermediate compounds, which then turns into a complex hydroxyether). 17-Alkoxide is easily converted into a complex hydroxyether after treatment with acid. Therefore, it is not generally found in the matrix product.

5-Cyano-hydroxyether, 5-cyano-7-acid and 17-alkoxide are new compounds which can be used as chromatographic markers and as intermediates to produce complex hydroxyether. They can be separated from the crude product in this stage of the Scheme 1 synthesis. Alternatively, they can be synthesized for use as markers or intermediate compounds. 5-Cyano-hydroxyether can be synthesized by reaction of a solution of the selected diketone of formula VI with a base such as alkoxide or amine, followed by separation of the resulting sludge. The preferred compound is 17-methyl who Idro-5β -cyano-11α,17-dihydroxy-3-oxo-17α-pregnan-7α,21, in primary forms, γ-lactone.

5-Cyano-7-carboxylic acid can be synthesized by reaction of the diketone of formula VI with a weak aqueous base, such as sodium acetate or sodium bicarbonate, followed by separation of the resulting residue. The obtained 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 by the reaction of a solution of complex hydroxyether of formula V with an alkoxide to obtain a mixture of 17-alkoxide and the corresponding complex hydroxyether. The obtained compound is preferably dimethyl-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone.

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

and hydroxyphenyl product corresponds to formula VA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA, a R1defined in formula V. Preferably, R3represents hydrogen.

The products of formula V are novel compounds which are mainly used as intermediates DL is producing compounds of the formula I, and especially of formula IA. Preferably, the compounds of formula V correspond to formula VA, where-a-a -- - - represent-CH2-CH2-, R3represents hydrogen, lower alkyl or lower alkoxy, and R8and R9taken together, represent the 20-spiroxamine ring:

The most preferred compound of formula V is Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone.

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

In a preferred embodiment of the invention, methanol and HCN are removed after the end of the period of reaction by distillation, with a mineral acid (such the AK hydrochloric acid or sulfuric acid) is added before the distillation, and water is added after distillation. The mineral acid may be added entirely in one piece, Paladino or continuously. In a preferred embodiment, the mineral acid is added continuously during the period of time from about 10 to about 40 minutes, and more preferably from about 15 to about 30 minutes. Similarly, water can be added to kubulau residue in one piece, Paladino or continuously. In a preferred variant of the invention, the concentrated reaction mixture after heating under reflux is cooled, and then add water. Before adding water, the mixture is preferably cooled to a temperature in the range from about 50°C to 70°C, preferably from about 60°C to 70°s, and more preferably up to about 65°C. Then, water is added, preferably continuously during the period of time from about 15 minutes to about 3 hours, and more preferably from about 60 minutes to about 90 minutes, maintaining approximately constant temperature. The product of formula V begins to crystallize from VAT residue as you add water. After adding water to the mixture of the diluted reaction mixture is support at the same temperature for about 1 hour, and then cooled to about 15°C for an additional period of time from about the olo 4 to about 5 hours. This mixture support at a temperature of about 15°during the period of time from about 1 to 2 hours. Maintaining the mixture at 15°With over a longer period of time leads to an increase in output of complex canoeiro in the mixture. This selection provides obtaining crystalline high quality product without the implementation procedures of extraction.

In accordance with another preferred method of selection of the product of formula V methanol and HCN are removed after the time of reaction by distillation, and the water and acid is added before distillation or distillation process. The addition of water before distillation simplifies operations, and gradually adding it to the distillation process allows you to maintain in Cuba, mainly, a constant volume. The product of formula V is crystallized from VAT residue in the distillation process. This selection provides obtaining crystalline high quality product without the implementation procedures of extraction.

In accordance with another alternative, the reaction solution containing the product of formula V can be suppressed by adding a mineral acid, such as 4n HCl, after which the solvent is removed by distillation. Removal of the solvent is also effective to remove residual HCN from the reaction PR the product. It has been found that purification of the compounds of formula V, where the compound of formula V is used as intermediate compounds in the process of getting epoxyoctane described above, needn't spend a lot of extraction solvent. In fact, often such extraction can be completely excluded. In the case when cleaning use solvent extraction, it is desirable to additionally carry out washing saturated salt solution and the alkaline washing. But when solvent extraction is not carried out, then rinse the salt saturated solution and alkaline leaching are also unnecessary. Exception procedures of extraction and leaching, mainly contributes to the increase in the productivity of this process, without, however, adversely affecting the yield and quality of the product, and also avoids the necessity of drying the washed solution using a desiccant such as sodium sulfate.

Raw 11α-hydroxy-7α-alkoxycarbonyl the product is again dissolved in a solvent for the next reaction stage of the process, which is a transformation of the 11-hydroxy group into a leaving group in the 11-position, resulting in a receive connection formula IV:

where: -a-a -, - - -, R3, R8and R9 defined above in formula XIII; R1defined in formula V, and R2is lower arylsulfonate, alkylsulfonate, acyloxy or halide. Preferably, 11α-hydroxy atrificial by reaction with a lower alkylsulfonates, allelochemical or anhydride of the acid, which is added to the solution containing the intermediate product of formula V. the Anhydrides of lower acids, such as anhydrides of acetic acid and anhydrides trihalomethanes acid such as the anhydride triperoxonane acid, can be used to obtain the corresponding leaving acyloxy groups. However, preferred are the halides of lower alkylsulfonic acid, and particularly preferred is methanesulfonanilide. Alternatively, 11α-hydroxy group can be converted to the halide by using a reaction of a suitable reagent, such as thienylboronic, thionyl chloride, sulfurylchloride or oxalicacid. Other reagents for the formation of esters 11α-sulphonic acid are taillored, benzosulphochloride and anhydride triftormetilfullerenov acid. This reaction is carried out in a solvent containing a halogen acceptor-hydrogen, such as triethylamine or pyridine. Can also be used inorganic bases such as potassium carbonate or the carb is NAT sodium. Initial concentration of complex hydroxyether of the formula V is preferably from about 5% to about 50 wt%. Tarifitsiruemih reagent is preferably present in a small excess. A particularly suitable solvent for this reaction is methylene chloride, but can also be used and other solvents, such as dichloroethane, pyridine, chloroform, methyl ethyl ketone, dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers, acetonitrile, toluene and tetrahydrofuran. The reaction temperature is determined mainly by the volatility of the solvent. In the case of methylene chloride, the reaction temperature is preferably from about -10°With up to about 10°C.

Hydroxyphenyl substrate of formula V preferably corresponds to the formula VA:

and the product corresponds to formula IVA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA, R1represents the lowest alkoxycarbonyl or hydroxycarbonyl, and R2defined in formula IV. Preferably, R3represents hydrogen.

The products of formula IV are novel compounds which are used primarily as intermediates for producing compounds of formula I, particularly compounds of the formula IA. Site is preferably, the compounds of formula V correspond to formula VA, where-a-a -- - - represent-CH2-CH2-, R3represents hydrogen, lower alkyl or lower alkoxy, and R8and R9together represent 20 spiroctan the first ring;

More preferred compound of formula IV is Metelitza-17α-hydroxy-11α-(methylsulphonyl)oxy-3-oxo-pregn-4-ene-7α,21, in primary forms, γ-lactone. If it is desirable presence leaving acyloxy group, the compound of formula IV is preferably 7 Metelitza-17-hydroxy-3-oxo-11α-(2,2,2-Cryptor-1 oksidoksi)-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone; or 7-methyl-11α-(atomic charges)-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone.

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

When you highlight a product of the formula IV or by receiving a reaction solution for converting intermediate compounds of formula IV in the intermediate compound of formula II, as described below, all stages of the extraction and/or leaching can be eliminated, and instead, the solution may be treated with ion exchange resins for the removal of acidic and basic impurities. This solution is first treated with anion exchange resin, and then cation exchange resin. Alternatively, the reaction solution may be first processed inorganic adsorbents, such as basic alumina or primary silicon dioxide, followed by rinsing with dilute acid. The main silica or basic alumina can be largely mixed with the reaction solution in the ratio of 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 using ion exchange resins or inorganic adsorbents processing can be carried out simply by suspension of resin or inorganic adsorbent of the reaction solution with stirring at room temperature and with the subsequent removal of resin or inorganic adsorbent by filtration.

In an alternative and preferred embodiment of the present izopet is of the compound obtained of the formula IV is isolated in crude form in the form of a concentrated solution by removing part of the solvent. This concentrated solution is directly used in the next stage of the process, in which of the compounds of formula IV are removed leaving 11α-group, resulting in a get complicated enefer formula II:

where: -a-a -, - - -, R3, R8and R9defined above in formula XIII, and R1defined in formula V. For the implementation of this reaction, the substituent R2the compounds of formula IV can be any leaving group, the removal of which is effective for the formation of a double bond between the 9 - and 11-carbon. The leaving group is preferably a lower alkylsulfonate or acyloxy Deputy, which is removed by reaction with acid or alkali metal salt. This can be used mineral acids, but preferred are lower alcamovia acid. In addition, the preferred reagent for this reaction is a salt of an alkali metal and used alanovoy acid. Especially preferably, this group contained mesilate, and a reagent for this reaction contained formic acid or acetic acid, or the alkali metal salt of one of these acids or other low alanovoy acid. If the leaving group is mesilate and remove the reagent is either acetic acid acetate sodium, either formic acid and potassium formate, then reached a relatively high ratio 9,11-olefin to 11,12-olefin. If in the process of removal of the leaving group is free water, there is a tendency to the formation of impurities, in particular, 7,9-lactone

(where: -a-a-, R3,- - - , R8and R9defined above in the formula (XIII), which are difficult to remove from the final product. Therefore, removal of water present in formic acid using acetic anhydride or other desiccant. The content of free water in the reaction mixture before the reaction should be maintained below about 0.5%, preferably below about 0.1 wt. -%, as determined by Karl Fischer analysis for water based on the weight of the total reaction solution. Although it is preferable that the reaction mixture was maintained almost in a dry condition, good results can be obtained when the water content of 0.3% of the mass. Thus preferably, the boot, the reaction mixture contains from about 4% to about 50 wt%. substrate of formula IV in alanovoy acid. Preferably, this mixture contains from about 4% to about 20% of the mass. salt of the alkali metal with acid. If the desiccant is used acetic anhydride, it is preferable that he attended the inratio from about 0.05 mole to about 0.2 mole per mole alanovoy acid.

It was found that the amount of by-product 7,9-lactone and 11,12-olefin in the reaction mixture will be relatively small, if the reagent for the elimination contains a combination triperoxonane acid, triperoxonane anhydride and potassium acetate, which is used as a reagent for the elimination of the leaving group and formation of complex enefer (9,11-olefin). Triperoxonane anhydride serves as a dewatering agent and must be present in an amount of at least about 3 wt. -%, more preferably at least about 15 wt. -%, and most preferably about 20% of the mass. in calculating the masses eliminating reagent containing triperoxonane acid.

In this stage of the synthesis Scheme 1, in addition to the 7,9-lactone, there is the presence of other impurities and by-products that can be used as intermediates for synthesis and as a chromatographic markers. New 4,9,13-triene complex tafira formula II (for example, 7-Metelitza-17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7α,21, in primary forms) was separated from the product solution by using chromatography. It is obvious that produced the amount of this compound increases with increasing reaction time for this stage of the synthesis. It has been suggested that this compound is formed by the protonation of the lactone and the received ion C17-Carbonia promotes migration angular methyl group of the C13 position. Deprotonation of this intermediate leads to the formation of 4,9,13-triens.

New 5-cyano-Δ11,12connection of complex tafira formula II (for example, 7-Metelitza-5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-in primary forms, γ-lactone) and a new 5-cyano-complex compound of tafira formula II (for example, 7-methyl-hydro-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-Yong-7α,21-in primary forms, 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 the synthesis Scheme 1.

New 17-epimer complex tafira formula II (for example, 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21-in primary forms, γ-lactone) was also isolated from the crude product by chromatography. It has been suggested that acidic reaction conditions, elimination can lead to racemization of chiral C17 center with obtaining 17-epimer complex enefer. 17-epimer can be synthesized by reaction of compounds of formula IV with a solution of potassium formate, formic acid and acetic anhydride, followed ejecta is m 17-epimer.

Although the solution of the crude product was not observed impurities, 11-ketone complex hydroxyether of formula V can be obtained by oxidation of 11-hydroxy corresponding complex hydroxyether a suitable oxidizing agent such as Jones reagent. Received 11-ketone is preferably 7 Metelitza-17-hydroxy-3,11-dioxo-17α-pregna-4-ene-7α,21, in primary forms, γ-lactone.

The alternative, leaving 11α-group of compounds of formula IV can be removed by obtaining complex tafira formula II by heating a solution of the 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 subjected to a reaction with alkenyl-alkanoate, such as isopropenylacetate, in the presence of acid, such as toluensulfonate acid or anhydrous mineral acid, such as sulfuric acid, with formation of ester 3-enol

the compounds of formula IV. Alternatively, an ester of 3-enol can be obtained by treating the compounds of formula IV with the acid anhydride and a base such as acetic acid and sodium acetate. Other alternative methods are the treatment of compounds of formula IV with ketene in the presence of acid to obtain the compounds of formula IV(Z). Interim connected to the e of the formula IV(Z) is then subjected to reaction with alkali metal formate or acetate in the presence of formic or acetic acid to obtain Δ 9,11-enolacetate formula IV(Y):

which can then be converted into a complex enefer formula II in an organic solvent, preferably alcohol, such as methanol, or by thermal decomposition of enolacetate or by its reaction with alkali metal alkoxide. The elimination reaction is highly selective for complex tafira formula II, preference is given 11,12-olefin and 7,9-lactone and this selectivity is preserved in the conversion process enolacetate in the exact location.

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

and the product corresponds to formula IIA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA, R1defined in formula V, and R3is preferably hydrogen.

If necessary, the connection formulas can be isolated by removing the solvent, dissolving the solid product in cold water and extraction with an organic solvent such as ethyl acetate. After the appropriate stages of washing and drying the product produce by removal of solvent for extraction. Then complex enefer dissolved in a solvent suitable for conversion into product Forms the crystals I. Alternatively, complex enefer can be isolated by adding water to the concentrated solution and filtration of the solid product with a further preferred removal of the 7,9-lactone. The conversion of the substrate of formula II in the product of formula IA can be carried out by the method described in U.S. patent No. 4559332, which in its entirety is introduced in the present invention by reference, or more preferably by using the new reaction using halogenating stimulator, as described below.

In another embodiment, the present invention is a complex hydroxyether of formula V can be converted into a complex enefer formula II without isolating the intermediate compounds of formula IV. In this way complex hydroxyether dissolved in an organic solvent such as methylene chloride; and to the solution add or allerease agent, such as methanesulfonate or halogenation agent, such as sulfurylchloride. The resulting mixture was stirred and, if provided by halogenoalkane add the HCl acceptor, such as imidazole. This reaction is highly exothermic and, therefore, it should be at a controlled rate with full cooling. After addition of the base the resulting mixture is heated to a moderate temperature, for example from about 0°s up to matnog temperature or slightly above, and the reaction is carried out usually within a period of time from about 1 hour to about 4 hours. After completion of the reaction the solvent is removed, preferably under conditions of high vacuum (for example, from about 24" to about 28" Hg(inches of Hg), from about -10°C to about +15°S, more preferably from about 0 to about 5°C, concentration of the solution and remove excess base. Then, to turn into a complex enefer, the substrate is again dissolved in an organic solvent, preferably in a halogenated solvent such as methylene chloride.

Reagent for the elimination of the leaving group is preferably prepared by mixing organic acids, salts of organic acids and desiccant, preferably formic acid, a formate of an alkali metal and acetic anhydride, respectively, in a dry reactor. The addition of acetic anhydride is accompanied by heat and leads to the separation, and therefore the rate of addition must be properly regulated. To stimulate the removal of water the temperature of this reaction, the support is preferably in the range from about 60 to about 90°and most preferably from about 65 to about 75°C. Then the specified reagent is added to a solution of the product compound IV to implement the elimination reaction. After a period of time from Ocala to about 8 hours, the reaction mixture is preferably heated to a temperature of at least about 85° With, but preferably not higher than about 95°With, before full removal of volatile distillate, and then further heated until the reaction is completed, usually within from about 1 to about 4 hours. The reaction mixture is cooled and, after separation by standard methods of extraction, this complex enefer can be allocated, if necessary, by evaporation of solvent.

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

In accordance with another alternative method of transformation of compounds of formula V in a complex enefer formula II without isolating the compounds of formula IV 11α-hydroxy group of complex hydroxyether formula V is replaced by halogen, then complex enefer formula II is formed in situ due to thermal dehydrohalogenation. Replacement of the hydroxy group by halogen carried out by reaction what sulfurylchloride, preferably sulphuraria, when cooled in the presence of acceptor galgenwaard, such as imidazole. Complex hydroxyether dissolved in a solvent such as tetrahydrofuran, and cooled to a temperature of from about 0°up to about -70°C. add sulfurylchloride and the reaction mixture is heated to a moderate temperature, for example to room temperature over a period of time sufficient to complete the reaction elimination, usually within a period of time from about 1 to about 4 hours. The method of implementation of this variant of the invention not only allows you to combine the two stages into one, but also avoids the use of: halogenated reaction solvent; an acid (such as acetic acid) and desiccant (such as acetic anhydride or sodium sulfate). In addition, this reaction does not require the conditions of heating under reflux, and avoids the production of by-product, formed by the use of acetic acid as a drying agent.

In accordance with a particularly preferred embodiment of the present invention, diketonate compound of formula VI can be converted into epoxyoctane or other compound of formula I without isolation of any intermediate compounds in purified form. In accordance with the laws the AI with this preferred method the reaction solution, containing complex hydroxyether, quenched with a solution of a strong acid, cooled to room temperature, and then extracted with an appropriate solvent for extraction. Before extraction to the reaction mixture it is preferable to add an aqueous solution of inorganic salts, for example about 10% of the mass. saturated salt solution. The extract is washed and dried by azeotropic distillation to remove methanol solvent remaining after cleavage reaction of the ketone.

Then, the concentrated solution containing from about 5% to about 50 wt%. the compounds of formula V is subjected to a contact in a cold state with allermuir or alkylsulfonyl reagent with the formation of ester sulfonic acid or a complex ester of dicarboxylic acid. After completion of the reaction alkylsulfonate or carboxylation reaction solution was passed through a column of acid, and then with a basic ion exchange resins for the removal of basic and acidic impurities. After each pass the column was washed with the appropriate solvent, e.g. methylene chloride, to highlight her residual of ester sulfonic or dicarboxylic acid. The combined eluate and factions of the washing are combined and concentrated, preferably in vacuo, resulting in a gain concentrated the initial solution, containing ester sulfonic or dicarboxylic acid of the formula IV. Then the concentrated solution is subjected to kontaktierung with a dry reagent containing an agent effective for the removal of the leaving 11α-ester groups and removal of hydrogen with the formation of the 9,11-double bond. Preferably, the reagent for removal of the leaving group contains a solution of dry reagent formic acid/formate of an alkali metal/acetic anhydride, as described above. After completion of the reaction, the reaction mixture is cooled, and formic acid and/or other volatile components are removed under vacuum. The residue is cooled to room temperature, subjected to appropriate stages of washing, and then dried, resulting in getting a concentrated solution containing complex enefer formula II. This complex enefer can then be converted into epoxyoctane or other compound of formula I using the method described above, or the method described in U.S. patent No. 4559332.

In a particularly preferred embodiment of the invention the solvent is removed from the reaction solution under vacuum and the product of formula IV is distributed between water and an appropriate organic solvent, for example ethyl acetate. Then the aqueous layer was subjected to back extraction with an organic dissolved the LEM and the reverse extract is washed with an alkaline solution, preferably a solution of alkali metal hydroxide containing a halide of an alkali metal. The organic phase is concentrated, preferably in vacuo and get inevery product of formula II. This product of formula II can then be dissolved in an organic solvent, for example methylene chloride, and after that it can be subjected to reaction in the manner described in the patent 322 to obtain the product of formula I.

If the epoxidation reaction is trichloracetonitrile, how it was established, it is very important the choice of solvent, with a high degree preferred are halogenated solvents, and particularly preferred is methylene chloride. Solvent, such as dichloroethane and chlorobenzene, provide an ample output, however, much higher output gives the reaction medium containing methylene chloride. Solvents such as acetonitrile and ethyl acetate, basically, give insufficient output, 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 of the synthesis epoxyoctane can be implemented by using for the epoxidation reaction as Perak odnogo activator trichlorinated instead of trichloracetonitrile. In a particularly preferred method of this epoxidation carried out by reaction of the substrate of formula IIA with hydrogen peroxide in the presence of trichloroacetamide and the corresponding buffer. This reaction preferably is carried out at a pH ranging 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 pH having values outside these preferred limits.

The most suitable results are obtained using a buffer containing dailybeast, and/or using a buffer containing a combination of dailybeast and potassium phosphate in the respective ratios of from about 1:4 to about 2:1, and most preferably within about 2:3. Can also be used borate buffers, but they usually give a slower reaction conversion than the potassium phosphate or a mixture of K2HPO4/KH2PO4. Regardless of the buffer, it should provide a pH in the range specified above. Regardless of the whole buffer or pH accuracy that it can provide, it was observed that the reaction proceeds much more efficiently, if at least part of the buffer contains the ion of dibasic phosphate. Obviously, this ion can mainly be deposited in the form of a homogeneous catalyst p and the formation of the adduct or complex, contains the stimulant and hydroxyperoxides ion, the production of which, in turn, can play an important role in the mechanism of the epoxidation reaction. Thus, the quantitative requirement of dibasic phosphate (preferably on the basis of K2HPO4may be only a small concentration of catalyst. Basically, preferably To2HPO4was present in the 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 in methylene chloride, but the alternative can also be used and other halogenated solvents, such as chlorobenzene or dichloromethane. It was found that satisfactory results also give a toluene and a mixture of toluene and acetonitrile. Without pretending to any particular theory, it should be noted that the reaction proceeds more efficiently in the two-phase system in which the formed hydroperoxide intermediate compound is distributed in the organic phase with a low content of water and reacts with the substrate in the organic phase. Thus, the preferred solvents are those solvents whose solubility in water is low. Efficient allocation of toluene stimulation is associated by including another solvent, such as acetonitrile.

When the conversion of the substrates of the formula II in the products of formula I using toluene gives advantages, because these substrates are easily dissolved in toluene in contrast to those 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 separated by filtration. When carrying out reactions of transformation 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 highly preferred reagent, but can also be used and other trihaloacetic, such as triptorelin and chlorodifluoroacetate. Can also be used trihalogen-benzamid and other compounds having Allenova, alkenylphenol or alkenylphenol group (or another group, which promotes the transfer of the electron-acceptrules actions electron-acceptor groups on the amide carbonyl) between the electron-acceptor trihalomethanes group and the amide carbonyl. Can also be used heptafluorobutyrate, but with less favorable results. Typically, the peroxide activator may sootwetstwu the th formula:

R°C(O)NH2

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

where RPrepresents a group, which allows you to transfer an electron-acceptrules the effect of electron-acceptor groups on the amide carbonyl, and which is preferably chosen from arylene, alkenyl, quinil and(CX4X5)ngroups; X1X2X3X4and X5independently selected from halogen, hydrogen, alkyl, halogen-alkyl, cyano and zainoulline; and n is 0, 1 or 2; provided that when n=0, then at least one of X1X2and X3represents halogen; and if RPis -(CX4X5)nand n=1 or 2, at least one of X4and X5represents halogen. If any of X1X2X3X4and X5is not a halogen, it is preferably halogenated, and more preferably perhalogenated. Particularly preferred promoters are promoters, is that n=0, and at least two of X1X2and X3represent halogen; or such activators, in which RPis -(CX4X5)n; n=1 or 2; at least one of X4and X5represents halogen and the other of X4and X5represent halogen or perhalogenated; and X1X2and X3represent halogen or perhalogenated. Each of X1X2X3X4and X5preferably represents Cl or F, and most preferably Cl, although it can also be used mixed halides, i.e. perchlorates or perbromates and combinations thereof, provided that the carbon directly linked to a carbonyl amide, 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 substrate initially present. The hydrogen peroxide should be loaded in response at least in moderate excess, or it must constantly be added by passing the reaction of epoxidation. Although the reaction consumes only one to two equivalents of hydrogen peroxide per one mol of the substrate, but preferably, the hydrogen peroxide was loaded into the considerable excess relative to the original prisustvujem substrate and activator. Without pretending to any particular theory, it should be noted that the reaction mechanism involves the formation of adduct activator and peroxide anion and that this reaction is reversible shift of the equilibrium towards the opposite reaction and that it is therefore necessary to use a large initial excess hydrogen peroxide to undergo the reaction in the forward direction. The precise 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. Basically, the preferred temperature is from about 20°With up to about 30°but under certain solvents, such as toluene, the reaction can be advantageously carried out at a temperature of from about 60°C to 70°C. At a temperature of about 25°for the reaction typically requires 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, may be added an additional amount of activator and hydrogen peroxide.

At the end of the reaction cycle, the aqueous phase is removed, the organic reaction solution is preferably washed to remove water-soluble impurities, the Le which the product can be isolated by removing the solvent. Before removing the solvent of the reaction solution should be washed at least mild to moderately alkaline washing, for example with sodium carbonate. Preferably, the reaction mixture is washed successively: weak regenerating solution, such as weak (for example, about 3% wt.) a solution of sodium sulfite in water, an alkaline solution such as NaOH or KOH, preferably about 0,5h); an acidic solution, such as HCl (preferably about 1 h); end neutral washing, containing water or saline solution, preferably a saturated saline solution to minimize product loss. Before removing the reaction solvent can be added mainly another solvent such as an organic solvent, preferably ethanol, to ensure that this product can be highlighted by crystallization after distillation carried out to remove the more volatile reaction solvent.

It should be noted that this new method of epoxidation using trichloroacetamide or another new peroxide activator has implications far beyond the various schemes for obtaining epoxyoctane, and can actually be used for the formation of epoxides by means of olefinic double bonds in substrates a wide range of subject R the shares in the liquid phase. This reaction is particularly effective in unsaturated compounds, in which the olefins are terazosine and tizamidine, that is, RaRbC=CRcRdand RaRbC=CRcH, where Ra-Rdrepresent substituents that are not hydrogen. This reaction proceeds most quickly and completely, where the substrate is a cyclic compound with tizamidine a double bond or a cyclic or acyclic compound with a Tetra-substituted double bond. Examples of substrates for the reaction of epoxidation are Δ5,11-canrenone and the following substrates:

Since tizamidine and Tetra-substituted double bonds, the reaction proceeds more rapidly and more completely, it is particularly effective for the selective epoxidation on such double bonds in compounds, which may include other double bond, where the carbon atoms of the olefin are monosubstituted or even disubstituted.

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

In addition, it should be noted that this reaction can be used for the preferential reaction of epoxidation monosubstituted or even disubstituted double bonds, such as 11,12-olefin in a variety of steroid substrates.

However, since epoxidized mostly double bond with a higher degree of substitution, such as 9,11-olefin, with a high degree of selectivity, the method of the present invention is particularly effective for achieving high output and productivity in stages epoxidation in different reaction schemes are also described in this application.

It was shown that this improved method is particularly advantageous in its application for connection

by epoxidation connection

; and

getting connection

by epoxidation connection:

It was demonstrated many advantages of the method of the present invention, in which the reagent for transfer of oxygen in the PE the work epoxidation use trichloroacetamide instead of trichloroacetonitrile. System trichloroacetamide reagent has a low affinity for electron-deficient olefins, such as α,β-unsaturated ketones. This allows for the selective epoxidation of olefin with non-conjugate double bonds in the substrate containing both types of double bonds. In addition, in complex substrates, such as steroids, disubstituted and tizanidine olefins can be differentiated by the reaction. For example, good selectivity is observed when epoxydecane isomeric Δ-9,11 and Δ-11,12-compounds. In this case, 9,11-epoxide is formed with a minimum reaction isomer, containing Δ-11,12-double bond. In accordance with this, the yield of the reaction, the profile of the products and final product purity are mostly higher compared to reactions using trichloracetonitrile. In addition, it was found that significantly excess production of oxygen observed when using trichloracetonitrile, is minimized when using trichloroacetamide, giving greater credibility to this method of epoxidation. In addition, in contrast to the reactions stimulated by trichloroacetonitrile, the reaction using trichloroacetamide gives the minimum of the exothermic effect, which facilitates the regulation of the temperature prof is La reaction. According to the observations, it was found that in this reaction the effect of mixing is minimal, and the performance of the reactor is higher, and this reaction has additional benefits compared to the reaction carried out using trichloroacetonitrile. This reaction is more suitable for large-scale production than the response stimulated by trichloroacetonitrile. The selection of product and cleaning are simple. Oxidation Bayer-Villiger carbonyl functional group (stimulated peroxide conversion of the ketone to an ester) was not observed, as was revealed in the experiment using m-chloroperoxybenzoic acid or other perkiset. This reagent is inexpensive, easily accessible and high-tech.

In addition, in the raw product obtained in stage Scheme of synthesis 1, in which complex enefer formula II was converted into a compound of formula I, using chromatography was observed following connections:

(1) new 11α,12α-epoxide complex tafira formula II, for example 7-Metelitza-11α,12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone;

(2) new 4,5:9,11-diepoxide complex tafira formula II, for example 7-Metelitza-4α,5α:9α,11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α,21-dick is boxill, γ-lactone;

(3) a new 12-ketone complex tafira formula II, for example 7-Metelitza-17-hydroxy-3,12-dioxo-17α-pregna-4,9(11)-Dien-7α,21, in primary forms, γ-lactone;

(4) new 9,11-dihydroxy complex tafira formula II, for example 7-Metelitza-9α,11β17th trihydroxy-3-oxo-17α-pregna-4-ene-7α,21, in primary forms, γ-lactone;

(5) a new 12-hydroxy analogue complex tafira formula II, for example 7-Metelitza-12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21, in primary forms, γ-lactone; and

(6) new 7-acid compounds of the formula I, for example, 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid, γ-lactone.

These compounds are used as synthetic intermediates and/or chromatographic markers to obtain the compounds of formula I, in particular epoxyoctane.

It has been suggested that 11α,12α-epoxide complex tafira formula II is formed through impurity produced during the previous phase, in which the compound of formula IV is converted into a complex enefer formula II. This mixture was separated by chromatography and represents a complex Δ11,12-enefer. It is usually produced together with complicated Δ9,11-tefirom in the ratio of about 90:10 (Δ9,11-enefer:Δ11,12-enefer), although this relationship may ariyavamsa. As a result, oxylene complex Δ11,12-tafira in the process of converting complex tafira formula II to the compound of formula I is formed 11α,12α-epoxide.

4,5:9,11-Diepoxide complex tafira formula I produce using chromatography. It has been suggested that it results from sverhmotivirovanny complex enefer. In the crude product it is usually present in an amount of about 5% of the mass. or less, although this number may vary.

12-Ketone complex tafira formula II allocate using chromatography. It has been suggested that it is formed by allylic oxidation of complex enefer. In the crude product it is usually present in an amount of about 5% of the mass. or less, although this number can vary. Level 12-ketone detected in the crude product using trichloroacetonitrile as activator of hydrogen peroxide, was significantly higher than the level detected by using as an activator trichloroacetamide.

9,11-Dihydroxy-product complex tafira formula II allocate using chromatography. In the crude product it is usually present in an amount of about 5% of the mass. or less, although this number can vary. It has been suggested that it is formed by hydrolysis of the epoxide of formula I.

12-Hydroxy-is the product of complex tafira formula II allocate using chromatography. In the crude product it is usually present in an amount of about 5% of the mass. or less, although this number can vary. It has been suggested that it is formed by hydrolysis of the 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 obtain metabolite derived procarcinogen connection or the like, with improved properties such as increased solubility and absorption), which facilitates the introduction and/or efficiency epoxyoctane. 6-Hydroxy compounds of formula I (for example, 7-Metelitza-6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone) are new compounds which have been identified as a possible metabolite in rats. 6-Hydroxy-metabolite can be obtained from the corresponding ethyl ester enol (for example, 7-Metelitza-9α,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone). Ethyl ester enol compounds of the formula I can be obtained in accordance with the procedure described in R.M.Weier & L.M.Hofmann (J.Med.Chem. 1977, 1304), which is introduced in the present description by reference. Then ethyl enol ether is subjected to reaction with m-chloroperoxybenzoic acid and the floor shall indicate the corresponding 6-hydroxy-compound of formula I.

It is, furthermore, suggested that salts of monocarboxylic epoxyoctane, especially potassium and sodium, can serve as a suitable alternative to simplify the introduction of the compounds of formula I of the individual, which shows the introduction of the aldosterone antagonist. In mild basic conditions it is possible to carry out selective opening spirolactone compounds of formula I without hydrolysis C7-ester substituent to obtain the corresponding 17β-hydroxy-17α-(3-propionic acid)-analogue. These analogues with open-loop are more polar than their lactone analogues, and have a shorter retention time in the analysis using reversed-phase HPLC. Acidic conditions usually lead to the regeneration of the lactone ring.

In more severe conditions, spirolactone opens and complex C7 hydrolyzed with formation of the corresponding products, 7β-hydroxy-17α(3-propionic acid)-7-acid analogs of the compounds of formula I. These dicarboxylic acids have a shorter retention time in their analysis using reversed-phase HPLC. Acidic conditions (for example, processing of the diluted acid, such as 0.1-4M hydrochloric acid), usually lead to the regeneration of the lactone ring dicarboxylic acid.

A new method of epoxidation of the present invention t is aetsa highly effective as the final stage of the synthesis Scheme 1. In a particularly preferred variant of the invention, the entire process of Scheme 1 is as follows:

Scheme 2.

The second circuit of new reactions of the present invention (Scheme 2) started using canrenone or other substrate corresponding to formula XIII

where: -a-a -, - - -, R3, R8and R9defined above in formula XIII.

In the first stage of this method, the substrate of formula XIII is converted into a product of formula XII

using the scheme of the reaction of zenderoudi, basically similar to the circuit described above for the conversion of the substrate of formula VIII in the intermediate compound of formula VII. Preferably, the substrate of formula XIII corresponds to the formula XIIIA:

and adaminaby product corresponds to the formula XIIA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA. Preferably, R3represents hydrogen.

In the second stage of Scheme 2, the enamine of formula XII hydrolyzing with intermediate diketonato product of formula XI:

where: -a-a -, - - -, R3, R8and R9defined above in formula XIII using the reaction schemes is, basically similar to the circuit described above for the conversion of the substrate of formula VIII in the intermediate compound of formula VII. Preferably, the substrate of formula XII corresponds to the formula XIIA:

and diketonaty product corresponds to formula XIA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA. Preferably, R3represents hydrogen.

In addition, in accordance with reaction scheme 2 diketone of formula XI is subjected to reaction with the alkoxide of the alkali metal with the formation of maxidone or other product corresponding to the formula X,

where every one-And-A -, - - -, R3, R8and R9defined in formula XIII, and R1defined in formula V.

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

as an intermediate product corresponds to the formula HA:

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA, a R1defined in formula V. Preferably, R3represents hydrogen.

Then maxr is non other compounds of formula X are subjected to 9α -hydroxylation in a new way biological transformations and receive the products of formula IX:

where: -a-a -, - - -, R3, R8and R9defined above in formula XIII, and R1defined in formula V.

Microorganisms that can be used in this stage hydroxylation 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, Bacterium cyclooxydans ATCC 12673, Cylindrocarpon radicicola ATCC 11011, Nocardia aurentia ATCC 12674, Norcardia restrictus 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 figures 1 and 2. Especially preferred is a method, illustrated in figure 1.

Media for cultivation, which can be used in reactions of biological transformation, 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 derived; and from about 0.05% to about 5% of the mass. available phosphorus. Particularly preferred media is for cultivation are the following environments:

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. the halide of an alkali metal; and from about 0.05% to about 0.5% of the mass. yeast derivative, such as avtorizovanniy yeast or yeast extract; from about 0.05% to about 5% of the mass. phosphate salts, such as2HPO4; 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;

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

Fungi can be cultured in a nutrient medium on the basis of soy flour or peptone, whereas actinobacteria and eubacteria can grow in the environment based on soy flour (to which is added a 0.5%-1% of the mass. salts of carboxylic acids, such as formate, sodium biological transformations) or in broth Muller-Hinton.

Receipt 11β-hydroxyechinenone of maxidone by fermentation is discussed in Example 19C. Similar methods biological transformations can be used to obtain other starting compounds and intermediate compounds. In Example 19A described biological conversion of Androstenedione 11β-GI is roxiodragtodisc. In Example 19 (C) described the biological transformation maxidone 11α-hydroxylysine, Δ1,2-maxrenn, 6β-hydroxylysine, 12β-hydroxybenzene and 9α-hydroxybenzene. In Example 19D described biological transformation canrenone in Δ9,11-canrenone.

The products of formula IX are novel compounds which can be isolated by filtration, washed with a suitable organic solvent, e.g. ethyl acetate, and recrystallized from the same or a similar solvent. They are valuable mainly as intermediate compounds for obtaining the compounds of formula I, particularly compounds of the formula IA. Preferably, the compounds of formula IX corresponds to the formula IXA, where-a-a -- - - represent-CH2-CH2-, R3represents hydrogen, lower alkyl or lower alkoxy, and R8and R9together represent 20-spiroxamine ring:

In the next stage of the synthesis Scheme 2, the product of formula IX is subjected to reaction with a dehydrating reagent (suitable dehydrating agents, such as PhSOCl or ClSO3N, known to experts) to obtain the compounds of formula II

where: -a-a -, - - -, R3, R8and R9defined above in formula XIII, and R1defined in formula V. Prepact the tion, the compound of formula IX corresponds to the formula IXA

as an intermediate product corresponds to formula IIA

where every one-And-A -, - - -, R3, Y1, Y2and X are defined in formula XIIIA, and R1defined in formula V. Preferably, R3represents hydrogen.

In the final stage of this scheme of the synthesis of the product of formula II is converted into a product of formula I by epoxidation method described in U.S. patent 4559332; or preferably a new way epoxidation of the present invention described above.

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

Scheme 3.

In this case, the synthesis begins, based on the substrate corresponding to formula XX

where: -a-a - and R3defined above in formula XIII-B-B - defined in formula XIII, except that neither R6or R7are not part of a ring fused with ring D in the 16,17-positions, and R26represents lower alkyl, preferably methyl. Preferably, R3represents hydrogen. In the reaction of substrate of formula XX with ridom sulfone is formed of epoxy intermediate compound corresponding to fo the mule XIX:

where: -a-a -, - - -, R3and R26defined above in formula XX. R3preferably represents hydrogen.

In the next stage of the synthesis scheme 3, the intermediate compound of formula XIX turn into another intermediate compound of formula XVIII:

where: -a-a -, - - -, R3defined above in formula XX. R3preferably represents hydrogen. In this stage, the substrate of formula XIX is transformed into an intermediate compound of formula XVIII via reaction with NaCH(COOEt)2in the presence of a base in a solvent.

After heat treatment, the compounds of formula XVIII and processing water and alkali metal halide get decarboxylative intermediate compound corresponding to the formula XVII:

where: -a-a -, - - -, R3defined above in formula XX. R3preferably represents hydrogen.

This method of transformation of compounds of formula XX to a compound of formula XVII corresponds mainly to the method described in U.S. patent 3897417, 3413288 and 3300489, which in its entirety are introduced in the present description by reference. Although the substrates are different, however, reagents, mechanisms and conditions for the introduction of the 17-spirolactone parts are mostly the same.

The reaction prom which mediate the compounds of formula XVII with a dehydrating reagent receive another intermediate compound of formula XVI:

where: -a-a -, - - -, R3defined above in formula XX. R3preferably represents hydrogen.

Commonly used dehydrating reagents are dichlorodicyanoquinone (DDQ) and chloranil (2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, the dehydrogenation may be carried out by subsequent halogenation in the 6-position carbon, and then through the reaction of dehydrohalogenation.

Then, the intermediate compound of formula XVI is converted into a enamine of formula XVB:

where-a-a-, -B-b - and R3defined in formula XX. Preferably, R3represents hydrogen.

The reaction conversion is carried out by zenderoudi, basically as described above for the conversion of 11α-hydroxycodone formula VIII in the enamine of formula VII. Usually, the source of cyanide ions may be the alkali metal cyanide. The base is preferably pyrrolidin and/or tetramethylguanidine. Can be used and methanol solvent.

The products of formula XVB are new compounds which can be separated by chromatography. These and other new compounds of formula XV are intermediate compounds for preparing compounds of the formula I, especially of formulae IA. The compounds of formula XV correspond to the structure:

where-a-a -, - - -, R3, R8and R9defined in formula XIII. In the most preferred compounds of formula XV and formula XVB-a-a - and-B-To - represent-CH2-CH2-, and R3represents hydrogen.

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

where-a-a-, -B-b - and R3defined in formula XX. Preferably R3represents hydrogen.

Particularly preferred for the synthesis of epoxyoctane are those compounds of formula XIV, which are also covered by formula XIVB defined below.

The products of formula XIVB are new compounds which can be isolated by precipitation. These and other new compounds of formula XIV are intermediate compounds for preparing compounds of the formula I, especially of formulae IA. The compounds of formula XIV correspond to the structure:

where-a-a -, - - -, R3, R8and R9defined in formula XIII. In the most preferred compounds of formula XIV and XIVB-a-a - and-B-To - represent-CH2-CH2-, a R3represents hydrogen.

Then the compounds of formula XIVB converted into the compounds of formula XXXI, basically in the manner described above for the conversion is iceton formula VI in complex hydroxyether formula V. In this case, you must first select an intermediate compound XXXI:

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

where-a-a-, -B-b - and R3defined in formula XX, and R1defined in formula V. Preferably, R3represents hydrogen. Preferred compounds of formula XXXI are compounds covered by formula IIA. The compounds of formula XXXI is transformed into the compounds of formula XXXII by the method described above or described in U.S. Patent No. 4559332.

The 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'-hexadecagon-10β-,13'β-dimethyl-3',5,20'-dioxaspiro[furan-2(3H),17'β-[4,7]methane[17H]cyclopent[a]phenanthrene]5'-carbonitrile; and a 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'-tetradehydro-10'α,13'α-dimethyl-3'5 ,dioxaspiro[furan-2(3H),17'α(5 N)-[7,4]metheno[4H]-cyclopent[a]phenanthrene]-5'-carbonitrile. In the most preferred embodiment of the present invention the complete reaction Scheme 3 is as follows:

Scheme 4.

The first three stages of Scheme 4 are similar to the stages of the Circuit 3, i.e. the stage of obtaining the intermediate of formula XVII, based on the compounds according to the existing formula XX.

Then the intermediate compound of formula XVII epoxidized, for example by the method described in U.S. Patent No. 4559332, obtaining the compounds of formula XXIV:

where-a-a-, -B-b - and R3defined in formula XX. However, in a particularly preferred embodiment of the present invention, the substrate of formula XVII epoxidised on the 9,11-double bond using an oxidizing agent containing a peroxide activator type amide, most preferably trichloroacetamide, in accordance with the method described above in Scheme 1 for the conversion of complex tafira formula II in the product of formula I. the Terms and ratios of reagents for this reaction are mainly the same as described for the reaction of transformation of complex tafira formula II in epoxyoctane. Especially preferred compounds of formula XXIV are those compounds in which-a-a - and-B-B - are as defined for formula XIII, R3represents hydrogen.

It was found that the epoxidation reaction of the substrate of formula XVII can also give a very good yield using percolate, such as, for example, m-chloroperoxybenzoic acid. However trichloroacetamide reagent gives the best result for minimizing the formation of side product resulting from the oxidation of Bayer-Willie the EPA. This byproduct can be removed, but this requires the rubbing of the solvent, such as ethyl acetate followed by crystallization from another solvent, such as methylene chloride. Epoxy-compound of formula XXIV is subjected to dehydration with the formation of a double bond between the 6 - and 7-carbon by reaction with a dehydrating agent (oxidant)such as DDQ or chloranil, or using a sequential reaction of synthesized/dihydrobromide (or other reaction of halogenation/dehydrohalogenation with getting another new intermediate compounds of formula XXIII.

where-a-a -, - - -, R3, R8and R9defined in formula XX. Especially preferred compounds of formula XXIII are those compounds where-a-a - and-B-B - are as defined for formula XIII, a R3represents hydrogen.

Although direct oxidation is effective to obtain a product of formula XXIII, however, this reaction gives mainly low outputs. Therefore, the oxidation reaction is preferably carried out in two stages, first carry out halogenoalkane substrate of formula XXIV in C-6-position, and then spend dehydrohalogenation with the formation of 6,7-olefin. The halogenation reaction is conducted preferably with N-galagedara the practical reagent, such as, for example, N-bromosuccinimide. The reaction of the synthesized carried out in a suitable solvent, such as, for example, acetonitrile, in the presence of stimulator halogenation, such as benzoyl peroxide. This reaction proceeds effectively at temperatures ranging from about 50 to about 100°With, usually by heating under reflux in a solvent such as carbon tetrachloride, acetonitrile or mixtures thereof. However, to complete the reaction typically takes from 4 to 10 hours. After the reaction the solvent is evaporated and the residue is dissolved in water-immiscible solvent, such as ethyl acetate. The resulting solution was sequentially washed with a weak alkaline solution (such as bicarbonate of an alkali metal and water, or preferably a saturated saline solution to minimize loss of product, after which the solvent is evaporated and the residue is dissolved in another solvent (such as dimethylformamide), suitable for the reaction of dehydrohalogenation.

Suitable dehydrohalogenating reagent, for example 1,4-diazabicyclo[2.2.2]octane (DABCO), added to the solution together with the alkali metal halide such as LiBr, the solution is heated to a suitable reaction temperature, e.g. 60-80°and the reaction is carried out for several hours, usually from 4 to 15 hours to complete digitop the formation. If necessary, during the reaction cycle can be added an additional amount dihydrobromide reagent to bring the reaction to completion. Then the product of formula XXIII can be selected, for example by adding water to precipitate the product, which is then separated by filtration and, preferably, washed with some water. This product is preferably recrystallized, for example from dimethylformamide.

The products of formula XXIII, such as 9,11-epoxiconazol, are new compounds which can be isolated by extraction/crystallization. They are important as intermediate compounds for preparing compounds of the formula I, especially of formulae IA. For example, they can be used as substrates for producing compounds of formula XXII.

Using, basically, the method described above to obtain compounds of the formula VII, the compounds of formula XXIII is subjected to reaction with cyanide ion to produce new apoxyomenos compounds of the formula XXII:

where-a-a -, - - -, R3, R8and R9defined in formula XX. Especially preferred compounds of formula XXII are compounds in which-a-a - and-B-B - are as defined for formula XIII, R 3represents hydrogen.

The products of formula XXII are new compounds which can be isolated by precipitation and filtration. They are important as intermediate compounds for preparing compounds of the formula I, especially of formulae IA. In the most preferred compounds of formula XXII-a-a - and-B-To - represent-CH2-CH2-, and R3represents hydrogen.

Using, basically, the method described above to obtain compounds of formula VI, epoxyamine the compounds of formula XXII are made into new epoxydecane the compounds of formula XXI:

where-a-a -, - - -, R3, R8and R9defined in formula XIII. In the most preferred compounds of formula XXI-a-a - and-B-To - represent-CH2-CH2-, and R3represents hydrogen.

The products of formula XXI are new compounds which can be isolated by precipitation and filtration. They are important as intermediate compounds for preparing compounds of the formula I, especially of formulae IA. Especially 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-To - represent-CH2-CH2-, and R3to depict the place of hydrogen.

Using, basically, the method described above for obtaining complex hydroxyamine compounds of formula V from diketonate compounds of formula VI, epoxydecane the compounds of formula XXI is transformed into the compounds of formula XXXII:

where-a-a-, -B-b - and R3defined in formula XX, and R1defined in formula V.

As the reaction conversion of the diketone of formula V in a complex hydroxyether formula VI, 5-β-cyano-7-ester intermediate compound is also formed by reactions of epoxidation formula XXI in the compounds of formula XXXII. Both of these 5-β-cyano-7-ester intermediates can be isolated by treatment of the corresponding diketone alcohol, such as methanol, in the presence of a base such as triethylamine. Preferably, these intermediate compounds are obtained by boiling under reflux a mixture of the diketone in alcohol, such as methanol, containing from about 0.1 to about 2 equivalents of triethylamine per one mol of the diketone, for from about 4 to about 16 hours. The products isolated in pure form by cooling the mixture to about 25°With subsequent filtering. Selected intermediate compounds can be converted into compounds of formula XXXII by treatment with base, such as alkali metal alkoxide, will dissolve in the Le, preferably the alcohol, such as methanol. The use of alkoxide in alcohol gives the equilibrium mixture, similar to the mixture, which is formed in the case of processing the corresponding diketone of formula XXI under the same conditions.

In addition, in the crude product of the final stage of the method of Scheme 4 using chromatography was discovered complex 7β-ester compounds of formula XXXII (for example 7-Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7β,21, in primary forms, γ-lactone). The alkoxide and/or cyanide in solution react with each other : the transformation of complex 7α-ether in epimeno a complex mixture of 7α-ether and 7β-essential ephemera. Clean complex 7β-ether can be isolated from epimeres mixture by selective crystallization.

Preferably the compound of formula XXI is 4'S(4'α),7'α-9',11α-epoxyhexane-10β-,13'β-dimethyl-3',5,20'-dioxaspiro[furan-2(3H),17'β-[4,7]methane[17H]-cyclopent[a]-phenanthrene-5'-carbonitrile; a compound of formula XXII is 5'R(5'α),7'β-20'-amino-9,11β-epoxyhexane-10,13'-dimethyl-3',5-dioxaspiro[furan-2(3H),17'α-(5 N)-[7,4]mean[4H]-cyclopent[a]phenanthrene-5'-carbonitrile; and a compound of formula XXIII is 9,11α-epoxy-17α-hydroxy-3-ekspresy-4,6-diene-21-carboxylic acid, γ-lactone.

In a particularly preferred embodiment, we shall Otsego invention complete the reaction process Scheme 4 is the following:

Scheme 5.

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

where: -a-a -, - - -, R3defined above in formula XX.

The following microorganisms capable of 9α-hydroxylation of compounds of formula XXXV (such as Androstenedione).

where: -a-a -, - - -, R3defined above in formula XIII, with the formation of compounds of formula XXIX in conditions similar to the conditions described in Example 19C:

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 is converted into the product of formula XXVIII:

by reaction with triethylorthoformate, where: -a-a-, -B-b - and R3defined above in formula XX.

After the formation of compounds of formula XXVIII, these compounds are converted into the compounds of formula XXVII in the manner described above for the conversion of the substrate of formula XX to a compound of formula XVII. The compounds of formula XXVII have the following structure:

where: -a-a -, - - -, R3defined above in formula XX, a Rxpresent which allows any of the standard hydroxy-protective groups.

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

The method described above to obtain compounds of formula XVI, a compound of formula XXVII are oxidized to produce new compounds of the formula XXVI

where: -a-a-, -B-b - and R3defined above in formula XX.

Especially preferred compounds of formula XXIX, XXVIII, XXV and XXVI are compounds in which-a-a - and-B-B - are as defined for formula XIII, R3represents hydrogen.

The products of formula XXVI are new compounds which can be isolated by precipitation/filtration. They mainly can be used as intermediates for producing compounds of formula I, particularly compounds of the formula IA. Especially preferred compounds of formula XXVI are compounds in which-a-a - and-B-B - are as defined for formula XIII, R3represents hydrogen. In the most preferred compounds of formula XXVI, -a-a - and-B-To - represent-CH2-CH2-, and R3to depict the place of hydrogen.

The method described above for zenderoudi compounds of formula VIII, the new intermediate compounds of formula XXVI are made into new 9-hydroxyanisole intermediate compounds of formula XXV

where: -a-a -, - - -, R3defined above in formula XX.

The products of formula XXV are new compounds which can be isolated by precipitation/filtration. They are mainly used as intermediates for producing compounds of formula I, particularly compounds of the formula IA. Especially preferred compounds of formula XXVI are compounds in which-a-a - and-B-B - are as defined for formula XIII, a R3represents hydrogen. In the most preferred compounds of formula XXVI-a-a - and-B-To - represent-CH2-CH2-, a R3represents hydrogen.

The method described above to obtain diketonate compounds of formula VI, 9-hydroxyanisole intermediate compounds of formula XXV in turn diketonate the compounds of formula XIVB. It should be noted that in this case the reaction is effective for simultaneous hydrolysis enaminones patterns and dehydration in 9,11-provisions for the introduction of a 9,11-double bond. Then the compound of formula XIV is converted into a compound of formula XIII using the same stage, which is as described above in Scheme 3.

Preferably, the compound of the 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'-hexadecagon-10β-13'β-dimethyl-3',5,20'-dioxaspiro[furan-2(3H),17'β-[4,7]methane[17H]-cyclopent[a]phenanthrene]-5'-carbonitrile; a compound of formula XXV is 5'R(5'α),7'β-20'-aminohexanoate-hydro-9'β-hydroxy-10'α, 13'α-dimethyl-3',5-dioxaspiro[furan-2(3H),17'α(5'H)-[7,4]metheno[4H]-cyclopent[a]phenanthrene]-5'- carbonitrile; a compound of formula XXVI is 9α,17α-dihydroxy-3-ekspresy-4,6-diene-21-carboxylic acid, γ-lactone; and a compound of formula XXVII is 9α,17α-dihydroxy-3-oxoprop-4-ene-21-carboxylic acid, γ-lactone.

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

Scheme 6.

Scheme 6 shows a preferred method of obtaining epoxyoctane and other compounds corresponding to formula I, based on 11α or 11β-hydroxylation of androstendione or other compound of formula XXXV:

where: -a-a-, -B-b - and R3defined above in formula XIII, to obtain the intermediate compound corresponding to the formula XXXVI or its corresponding 11β-hydroxy-isomer

where: -a-a-, -B-b - and R3defined above in formula XIII.

Except for the choice of the substrate, this method 11α-hydroxylation carried out basically as described above for Scheme 1. 11α-hydroxylation of androstendione or other compound of formula XXXV is capable of the following microorganisms:

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, Aspergillus nidulans ATCC 11267, Beauveria bassiana ATCC 7159, Fusarium oxysporum ATCC 7601, Fusarium 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 12519 and ATCC 8685.

11β-hydroxylation of androstendione or other compound of formula XXXV is capable of 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-ene-3,17-dione or a different compound of formula XXXVI is transformed into a simple ether 11α-hydroxy-3,4-enol of the formula (101):

where: -a-a-, -B-b - and R3defined above in formula XIII, and R11denotes methyl or other lower alkyl (C1-C4) through reaction with etherification reagent, such as trialkylaluminium, PR is the presence of an acid catalyst. To implement this transformation 11α-hydroxysulfate acidified by mixing with acid, such as, for example, hydrate benzosulfimide acid or hydrate toluensulfonate acid in a solvent such as a lower alcohol, for example ethanol. Trialkylaluminium, preferably triethylorthoformate introduce gradually over a period of 5-40 minutes, maintaining the mixture in a cold condition, preferably at a temperature of from about 0°With up to about 15°C. the mixture is Then heated and the reaction is carried out at a temperature from 20°C to 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 within 2-6 hours until the reaction is completed. The reaction mixture is cooled, preferably to 0°-15°s, and more preferably at about 5°and the solvent is removed in vacuum.

Using the same reaction scheme, which was described above in Scheme 3 for the conversion of compounds of formula XX to a compound of formula XVII, 17-spirolactone part of the formula XXXIII is introduced into the compound of formula 101. For example, the substrate of formula 101 may be subjected to reaction with ridom sulfone in the presence of a base such as an alkali metal hydroxide, in a suitable solvent, such as DMSO, to obtain the intermediate is of the compounds of formula 102:

where: -a-a-, R3, R11and At defined above in formula 101. The intermediate compound of formula 102 is then subjected to reaction with complex fluids malonic acid in the presence of alkali metal alkoxide to obtain a five-membered spirolactone ring and the intermediate compounds of formula 103:

where: -a-a-, R3, R11and At defined above in formula 102, and R12is1-C4alkyl, preferably ethyl. And finally, the compound of formula 103 in a suitable solvent, such as dimethylformamide, heated in the presence of a halide of an alkali metal, arseplay, however, alkoxycarbonyl group and receiving an intermediate compound of formula 104:

again-a-a-, R3, R11and At defined above in formula 102.

Then the ether compound 3,4-enol 104 is converted into a compound of formula XXIII, i.e. the compound of formula VIII, in which R8and R9taken together, constitute a part of the formula XXXIII. This stage of the oxidation is carried out, mainly, in the same way, which was carried out by phase oxidation for the conversion of compounds of formula XXIV in the intermediate compound of formula XXIII in the scheme of the synthesis of 4. Direct oxidation can be carried out using such a reagent is, as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or tetrachlorobenzoquinone (chloranil); or, preferably, two-stage oxidation is carried out first by synthesized, for example using N-halogen-brainwasher agent, such as N-bromosuccinimide (NBS) or 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), and then by dehydrobrominated using a base such as DABCO, in the presence of LiBr and at elevated temperatures. If for synthesized using NBS, for the conversion of ester 3-enol in the exact location should also be used acid. DBDMH, ion, and not of free radical pomeroyi reagent, is itself effective for the synthesized and conversion of the enol ether in the exact location.

Then the compound of formula VIII in turn epoxyoctane or other compound of formula I in the stages described above for Scheme 1.

Each of the intermediate compounds of formula 101, 102, 103 and 104 is a new connection, which can be mainly used as intermediate compounds for obtaining epoxyoctane or other compounds of formulas IA and I. In each of the compounds of formula 101, 102, 103 and 104-a-a - and-B-To - represent-CH2-CH2-, a R3represents hydrogen, lower alkyl or lower alkoxy. R3preferably represents hydrogen. Most preferably the compound of formula 101 is the 3-ethoxy-11α -hydroxyandrost-3,5-Dien-17-one; compound of formula 102 is a 3-toxisperma[androst-3,5-Dien-17β,2'-oxiran]-11α-ol, a compound of formula 103 is atingido-3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-in primary forms, gamma-lactone and a compound of formula 104 is a 3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid γ-lactone.

In a particularly preferred embodiment of the invention the entire process Circuit 6 is as follows:

It has been suggested that epoxyoctane and other compounds corresponding to formula I, can be also obtained from 11β-hydroxyandrostenedione or other compounds of formula XXXV, which were 11β-gidroksilirovanii. In other words, epoxyoctane and other compounds corresponding to formula I, can be obtained by the General method presented in Scheme 6 using either α-gidrauxilirovannogo substrate of formula XXXV or the β-gidrauxilirovannogo substrate.

Scheme 7.

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

where: -a-a-, R3and In particular is trading above in formula XIII; D-D represents-CH2-CH2- or-CH=CH-; and each of R13, R14, R15and R16independently selected from hydrogen or C1-C4the alkyl.

R3preferably represents hydrogen.

In the first stage of synthesis of 11α-hydroxyandrostenedione or other compounds of formula XXXVI is produced by a biological conversion of compounds of formula XXXVII. How such biological prevrasheniya carried out mainly by the method described above for 11α-hydroxylation canrenone (or other substrate of formula XIII).

In the synthesis of 11α-hydroxyandrostenedione 4-androsten-3,17-dione is first produced by a biological conversion of compounds of formula XXXVII. This is the original biological transformation can be carried out by the method described in U.S. patent No. 3759791, which in its entirety is introduced into the present description by reference. Then 4-androsten-3,17-dione in turn 11α-hydroxyandrostenedione mainly by the method described above for 11α-hydroxylation canrenone (or other substrate of formula XIII).

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

It has been suggested that epoxies is non other compounds corresponding to the formula I can also be obtained in accordance with the General method presented in Scheme 7, if the product of biological transformations β-sitosterol or other compounds of formula XXXVIII is 11β-hydroxyandrostenedione or other compounds of formula XXV, which were 11β-gidroksilirovanii. In other words, epoxyoctane and other compounds corresponding to formula I, can be obtained by the General method presented in Scheme 7, if the biological transformation β-sitosterol or other compounds of formula XXXVIII results or α-gidrauxilirovannogo substrate of formula XXXV or the β-gidrauxilirovannogo substrate.

Scheme 8.

A significant difficulty in the synthesis epoxyoctane and related compounds is necessary stereoselective introduction α-alkoxycarbonyl substituent at the 7-carbon, without causing undesirable modifications in other parts of the steroid structure. In accordance with the present invention it was found that the method is effective synthesis for the introduction of 7α-alkoxycarbonyl Deputy comprises the following stages: (i) initial zenderoudi 7-carbon steroid, (ii) hydrolysis of 7-lanosterol with the formation of a mixture of 7α-carboxylic acid and 7β-carbon to the slots steroids, (iii) formation of 5,7-lactonitrile 7α-carboxylic acid of the steroid, and (iv) division 7β-carboxylic acid of the steroid from 5,7-lactonitrile. In the indirect base reactions, ring opening, which occurs between 5,7-lactoserum and alkylating reagent, get right 7α-alkoxycarbonyl steroid.

In line with this, the process Circuit 8 is usually aimed at obtaining 3-keto-7α-alkoxycarbonyl-substituted Δ4,5-steroid, involving the reaction of an alkylating reagent of 3-keto-4,5-dihydro-5,7-lakestream substrate in the presence of a base. The lactone substrate is replaced by a keto group at the 3-position carbon and, in addition, contains part:

where(5) is a 5-carbon, and(7) represents the 7-carbon of the steroid structure of the substrate. The transformation of the 5,7-lactone 7α-alkoxycarbonyl preferably carried out by reaction with alkylhalogenide in the presence of a base. Alkylhalogenide reagent is preferably iodide, and most preferably methyliodide.

In addition, in accordance with the present invention was developed predominant way to obtain 4,5-dihydro-5,7-lactonitrile compounds described above. In this way 3-keto-Δ4,5-7α-tiananamen steroid substrate is converted into 7-carboxylic acid, as the acid, in turn, is subjected to reaction with trialkylaluminium in the acidified solvent, a lower alcohol, to obtain 5,7-lactone. Reaction with orthoformate esters promotes the conversion of 3-keto group in the 3-acyclic or cyclic ketal-5,7-lactone (it should be noted that first formed lactone). Preferred 3-ketal-5,7-lactone is a 3-dialcentral-5,7-lactone. More preferably the alkyl portion of the alcohol solvent is the same as the alkyl part of alkoxy groups orthoformiate (and most preferably all of them are stands)because: alkoxy part Catala can occur either from orthoformiate or alcohol; mixed ketals are not preferred; and 3-dimethoxy is preferred. If ketal is atelectases, the alkyl portion of the alcohol solvent is not necessarily the same as the alkyl part of alkoxy groups orthoformiate. 3-ketal-5,7-lactone easily hydrolyzed with the formation of 3-keto-5,7-lactone, crystalline compound that can be easily cleaned. Since the reaction of lactonization exposed only 7α-carboxylic acid provides full stereospecificity. 7β-Acid can then be removed from the reaction mixture as a salt, for example by processing 7β-weak acid the base, such as sodium bicarbonate.

7-Cyano-substrate for the production of 5,7-lactone can be obtained in a known manner. For example, the substrate, unsubstituted 7-position carbon, may be subjected to reaction with a slight excess cyanide ion, preferably from about of 1.05 to about 1.25 equivalents per equivalent of substrate in a slightly acidified solution containing a solvent mixture of water/DMSO. Preferably, the reaction mixture consisted of a carboxylic acid, for example about one equivalent of acetic acid per equivalent of substrate. This forms 7αand 7β-CN-isomer, and 7α-isomer is the major isomer. 7α-Cyano steroid can be selected in the standard way. This additional receipt can be used and other methods known to experts.

Basically, in accordance with Scheme 8, 5,7-lactone can be formed from the intermediate 7-carboxy-compounds (which itself is obtained by hydrolysis of the intermediate 7-cyano-compounds), which is substituted in the 17-position or a keto-group, or R8or R9where R8and R9defined above, and which has an aliphatic, olefinic, epoxy or hydroxy-substituted configuration at C-9 and C-11

where-a-a-, -B-b - and R3defined you is e, R80and R90are the same as R8and R9or R80and R90taken together form a keto, R18defined below for the Circuit 9, a-E-E - select from

The compound of formula XLII is then transformed into a 7α-alkoxy-carbonyl;

In each of the connections, XL, XLI, XLII and XLVIII R80and R90taken together preferably comprise keto or

where Y1, Y2X and C (17) defined above, and most preferably, if R80and R90taken together, constitute

R3preferably represents N, R1preferably represents methoxycarbonyl, and-And-And - and-In-In - preferably represent-CH2-CH2-. It should be noted that these reactions can also be carried out with the 3-keto group is protected by its transformation in every essential or catalog form and maintain it in this form throughout the sequence of reactions. Alternative methods of Scheme 8 involve the use of various intermediates, HC is tihamah formula XLI and XLII, defined above.

It should be noted that the reagent used for the formation of a 5,7-lactone of 3-keto-Δ4,5-7-carboxylic acid in Scheme 8 is trialkylaluminium, the same reagent used for the conversion of 11α-hydroxyandrostenedione in intermediate 3-enlever-3,5-Dien-11α-hydroxy-connection 101 of Scheme 6, it is evident that the way in which the Scheme of reactions 8, zavisit from substitution at C-7. In the reaction with orthoformate in the presence of N+formed intermediate carbonium ion with carboxyl in position C-7, and a positive charge when the balance between C-3 and C-5. After loss of a proton From the-3-ion Carbonia gives compound of formula 101, and p-5-ion Carbonia gives the lactone. In the case of the hydrogen at C-7 obviously, is mostly formed 3,5-diene-3-alkoxy (enol ether) due to conjugation of double bonds. If 7α-CO2gamestates at C-7, C-5-ion Carbonia is captured carboxy and formed 5,7-lactone. At this stage, the 3-keto group mainly develops in Catal that contributes to the completion of the reaction.

Preferred variants of the Circuit 8 is described below in Schemes 9 and 10.

Scheme 9.

Scheme 9 begins with the use of the same substrate that was used in figure 4, i.e. the compounds of formula XX. This substrate is first oxidized to connected the formula I;

where: -a-a-, R3and At defined above in formula XIII.

The oxidation reaction is carried out in accordance with any of the reaction schemes described above for the conversion of compounds of formula XXIV in the intermediate compound of formula XXIII in the synthesis Scheme 4. Using the methods described for scheme 8, the compound of the formula In turn 7-cyano-intermediate compound of the formula:

where-a-a-, R3and In defined in formula XIII. Then the compound of formula transformed into a 5,7-lactone of the formula D:

where-a-a-, R3and In defined in formula XIII, and R17is1-C4alkyl, using trialkylphosphates reagent used previously in Scheme 6. 5,7-Lactone of formula D is readily separated from unreacted compound 7-β-COOH, for example by removing the acid by washing with bicarbonate, and thereby create the desired stereochemical With-7-patterns and prevent epimerization in subsequent reactions are carried out in basic conditions. Etherification of the lactone by reaction with alkylhalogenide as described in Scheme 8, gives a complex Inferno intermediate compound of formula II.

Continuing the synthesis according to Scheme 9, the compound of formula D is converted into a compound of formula II. With what ispolzovaniem 3-keto group, secure by turning in ketal, 20-spiroxamine group of formula XXXIII selectively injected into the 17-position in accordance with the reaction scheme described above for Scheme 3 and 6 (see above), and obtain the connection formula:

Since the 3-ketone is protected, the conditions of hydrolysis can be selected so that they are optimal for the impact of the 17-ketone without the formation of by-products by reaction at the 3-position. After hydrolysis of 3-catalogo the compounds of formula E in the structure of 3-keto group of the formula F

the last intermediate compound is subjected to reaction with alkyllithium in the presence of a base Schema transformations 8 with the intermediate complex tafira formula II. And finally, the last intermediate connection in turn epoxyoctane or other compound of formula I using any of the methods described above for scheme 1.

Scheme 9 has advantages not only because of the possibility to control the stereochemistry reported 5,7-lactone intermediate connection, but it also provides additional benefits, which is the ability to use the conditions of hydrolysis over a wide range without affecting the 17-spirolactone.

Like the reactions of other schemes for the synthesis of this image is etenia reaction Scheme 9 can be used for the conversion of substrates, different from the substrates, specifically described above. For example, the conversion of 3-keto - or 3-ketal-7-cyano-steroids of the 3-keto - or 3-ketal-5,7-lactone, or the conversion of 3-keto - or 3-ketal-5,7-lactone 7α-alkoxycarbonyl can be carried out using compounds substituted at the 17-carbon groups R8and R9defined above, or, more preferably, Deputy formula:

where X, Y1and Y2defined above, and(17) mean 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 with the use of 17-keto-substrates and in accordance with the specific reaction scheme described above for the introduction of the 17-spirolactone and 7α-alkoxycarbonyl 3-keto-Δ9,11-steroid.

The lactones of formula D, E and F are new compounds which can be used to obtain epoxyoctane and other compounds of formulas I and IA in accordance with the Scheme of the synthesis of 9. In these compounds-a-a - and-B-In - preferably represent-CH2-CH2-, a R3represents hydrogen, lower alkyl or lower alkoxy. Most preferred is a compound of formula D, in which R17represents methoxy.

In particularly preferred in which the version of the invention the entire process Circuit 9 proceeds as follows:

Scheme 10.

The circuit 10 is the same as Scheme 9 up to the formation of intermediate 7-cyano compounds of formula C. In the next stage Circuit 10 7-cyano-steroid is subjected to reaction with trialkylaluminium in alkanolov solvent, preferably triethylorthoformate in methanol with simultaneous protection of 3-keto and 17-keto group by making the first connection to the enol ether, and the last in Catal. Then 7-cyano-group restore to 7-formyl-for example, by reaction with a hydride dialkylamide, preferably a hydride diisobutylaluminum, resulting in a receive connection formula 203:

where-a-a-, R3and At defined above in formula XIII, a R18is1-C4alkyl.

Before the protection of the keto-groups, as described above, recovery warn using hydride dialkylamide. Then the intermediate compound of formula 203 is subjected to reaction with dilute aqueous acid for the selective hydrolysis of the 17-Catala in the presence of excess alcohol (R19OH), resulting in a gain of the intermediate compound of formula 204:

where R19selected from lower alkyl (preferably C1-C4), or the group R193-state who form a cyclic O,O-oxyalkylene Deputy at the 3-carbon. Polyacetal [204], in addition, protect by processing alkanols (R19OH) in the presence of an anhydrous acid to obtain an intermediate compound of formula 205:

where: -a-a -, - - -, R3and R19defined above, and R20is1-C4alkyl.

17-Spirolactone part can then be entered in accordance with the reaction stages described above for Schemes 3 and 6, which is carried out in the following sequence:

where-a-a -, - - -, R3, R19and R20defined above, a R25is1-C4alkyl.

After that, the 3-position is removed protection by standard hydrolysis with the re-introduction of 3-keto-group and 5.7-polyacetale and get another intermediate compound corresponding to the formula 209:

where-a-a-, -B-b - and R3defined above.

Then 9,11-epoxy part administered by any of the methods described above for converting compounds of the formula II into compounds of the formula I. In the oxidation reaction conditions of the epoxidation Polyacetal partially transformed into a 5,7-lactone, the result of which is produced by another intermediate Obedinenie, corresponding to the formula 211:

where: -a-a-, -B-b - and R3defined above.

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

where: -a-a-, -B-b - and R3defined above, is easily oxidized by standard methods to obtain the compounds of formula 211. And, finally, the intermediate compound of formula 211 in turn epoxyoctane or other compound of formula I by the method described in Scheme 8 for the conversion of the 5,7-lactone 7α-alkoxy-carbonyl compound. Thus the whole Scheme 10 proceeds as illustrated below; however, it should be noted that at least the following stages can be carried out in situ without isolation of intermediate compounds. In General, the synthesis Circuit 10 is as follows:

As in the case of Scheme 9, the reactions described above for the Circuit 10, have important advantages, especially from the point of view of economy of way; and, at the same time, the new reaction Scheme 10 also have more General application in relation to the substrates, which are not substrates specifically described above. For example, the introduction of the 7-formyl group in the ester 3-enol steroid, protection of the resulting ester of 7-formyl-Δ-5,6-3,4-enol, hydrolysis OBR is using 5,7-Polyacetal and subsequent release can be performed on steroids, substituted in the 17-position by a group R8and R9defined above, or, more specifically, Deputy formula:

where each of X, Y1, Y2and(17) defined above.

Alternative methods of Circuit 10 include the use of various intermediate compounds covered by formula A203-a, respectively, described above. Each of the intermediate compounds of formula A-A is a new connection that can be used to obtain epoxyoctane and other compounds of formula I and IA in accordance with the Scheme of the synthesis of 10.

In a particularly preferred embodiment of the invention, the entire process Circuit 10 is as follows:

Based on several schemes illustrated above, it should be noted that the reaction stage, selected for use in the methods of the present invention provide considerable flexibility in the production epoxyoctane and related compounds. Key distinguishing features of these methods are, inter alia: (a) biological conversion of substrate, such as canrenone, Androstenedione or β-sitosterol 11αor 9α-hydroxy-derivative (concurrent transformation β-sitosterol 17-keto-structure; (b) the introduction of a 9,11-double the ligature by dehydration of compounds containing either 11αor 9α-hydroxy group, followed by the introduction of epoxy groups by oxidation 9,11-double bond; (C) attaching 7α-alkoxycarbonyl through the formation of enamine, hydrolysis of the enamine with the formation of the diketone and the reaction of the diketone with an alkoxide of an alkali metal; (d) education 20-spirostanol rings in 17-position; (e) education 5,7-lactone and etherification of this lactone with the formation of 7-alkoxycarbonyl; (f) protection of the 3-ketone by transformations in ether 3-enol or 3-ketal through a series of transformations in other provisions (including education 20-spirostanol rings in 17-position). With some limitations, these four-stage process (b)-(d) can be performed in almost any order. Stage of the process (e) and (f) comparative adaptability. They provide a way of obtaining epoxyoctane and other compounds of formula I and were greatly simplified in comparison with the method described in U.S. patent 4559332. In addition, they provide important advantages in terms of productivity and output.

In the descriptions of the reaction scheme shown above, synthesis, isolation and purification of the reaction products can be mainly carried out by methods well known in the art. Unless specifically indicated, the conditions, solvents and reagents is the fast or standard, either they don't have exceptionally critical value, or both. However, some of the specific procedures specifically described above have advantages, which provide the increase in total output and/or productivity of the various stages of the process and diagrams of this process, and/or high quality intermediate and final 9,11-epoxy-steroidal products.

Effective use of 20-pyroxenoid compounds produced in accordance with the present invention, is described in U.S. patent 4559332 (Grob), which in its entirety is introduced into the present description by reference.

20-Spiroxazine compounds obtained in accordance with the present invention, are distinguished by their good biological properties, and therefore are valuable pharmaceutically active ingredients. For example, they have a strong aldosterone-antagonistic action, which is that they reduce and normalize excessive accumulation of sodium and excretion of potassium caused by aldosterone. Therefore, they act as a potassium-retaining diuretics and may have important therapeutic applications, for example for the treatment of hypertension, congestive heart failure or cirrhosis of the liver.

Known 20-spiroxamine derivatives with aldosterone-antagonistic action, see, nab is emer, Fieser &Fieser: Steroids; p.708 (Reinhold Publ.Corp., New York, 1959) and description of the United Kingdom patent No. 1041534; also known 17β-hydroxy-21-carboxylic acids and their salts with similar activity, see, for example, U.S. patent No. 3849404. However, compounds of this type, used up to the present time as medicines, have significant disadvantages, namely that they always have some sexualna-specific activity, which sooner or later causes adverse effects in normal long-term therapy. Particularly undesirable are adverse effects that can be attributed antiandrogenna activity known antialdosterone drugs.

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

EXAMPLE 1.

Culture on the sloped agar were obtained with the use of media for culturing described in Table 1

Table 1.
YPDA (environment for crops on sloping agar and cups)
yeast extract20 g
peptone20 g
glucose20 g
agar20 g
distilled water, PT. number

- pH of 6.7

- bring to pH 5 by adding 10% wt./about H3RHO4
to 1000 ml
Distribution:

for beveled environments:

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

For producing cultures of the first generation of the colony of Aspergillus ochraceus 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 environments, which were obtained as described above. These beveled environment were incubated for seven days at 25°C, after which he appeared superficial culture, which was a white fluffy mycelium. The reverse side was painted in orange color in its lower part and the upper part is yellow-orange color.

Crops first generation suspended in a sterile solution (4 ml)containing nonionic surfactant tween 80 (3% wt.), and 0.15 ml aliquots of this suspension was used for inoculation of crops of the second generation, which was obtained with the use of media for culturing described in Table 2.

Table 2.
Environment for crops second generation standard beveled environment
extract of malt20 g
Peptone1 g
Glucose20 g
Agar20 g
distilled water, dotconnect

- pH of 5.3

- distribution 1.5-ml tubes (180×18 mm)

- sterilization at 120°C for 20 minutes
to 1000 ml

Crops of the second generation were incubated for 10 days at 25°With the result that received heavy mass of spores Golden color; the reverse side has been painted in a brown-orange color.

There was obtained a protective environment, having the composition indicated in Table 3.

Table 3.
Protective environment
Separated milk10 g
Distilled water100 ml
In a 250 ml flask containing 100 ml of distilled water was added to the separated milk at 50°C. Sterilized at 120°C for 15 minutes. Cooled at 33°C and used before the end of the day.

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

To obtain uterine crops suitable for large-scale fermentation, one aliquot liofilizovannyh culture, which was obtained by the method described above, suspended in distilled water (1 ml) and 0.15 ml aliquots of this suspension was used for inoculation beveled cultures, which were obtained with the use of media for culturing having the composition specified in Table 2. These uterine crops were incubated for seven days at 25°C. after incubation the culture grown on stubble agar, was stored at 4°is.

To obtain standard beveled culture culture obtained from the mother culture on the sloped agar, suspended in a sterile solution (4 ml)containing tween 80 (3% wt.), and the resulting suspension was distributed in 0.15 ml aliquot on stubble crops that were covered by the media for cultivation described in Table 2. Standard crops can be used for inoculation of primary seed flasks for laboratory or industrial fermentation.

For preparation of primary cultures in the seed flask culture from the standard crops, which were obtained as described above were removed and suspended in a solution (10 ml)containing tween 80 (3% wt.). 0,1-Aliquot of the resulting suspension was introduced into a 500 ml flask with a septum containing medium for cultivation, having the composition indicated in Table 4.

Table 4.
(for primary culture and transformation in the flask and in a round-bottom flask)
Glucose20 g
Peptone20 g
yeast autolysate20 g
distilled water, dotconnect

- pH of 5.2

- bringing to 5.8 by adding 20% NaOH

- distribution in 500 ml number of the e partition, 100 ml

the distribution in 2000 ml round-bottom flasks with baffles (3 partitions), 500 ml

- sterilization at 120°C for 20 minutes

pH after sterilization, about 5.7

Seed flask was incubated on a rotating shaker (200 rpm, displacement of 5 cm) for 24 hours at 28°C, resulting in a received culture in the form granulating mycelium pellets having diameters of 3-4 mm After evaluation under the microscope it was found that the seeding culture is a pure culture has cinematically the growth of hyphae and large, well twisted into a spiral. the pH of the suspension amounted to 5.4 and 5.6. PMV was 5-8%, as determined by centrifugation (3000 rpm × 5 min).

Culture for transformation in the flask was obtained by inoculation of media for culturing (100 ml)having the composition indicated in Table 4, in the second 500 ml shaker flask containing biomass (1 ml) from a flask with a seed culture. The resulting mixture was incubated on a rotating shaker (200 rpm, displacement of 5 cm) for 18 hours at 28°C. After exploring the culture, it was found that it contains granulomatosis mycelium pellets with a diameter of 3-4 mm After evaluation under the microscope it was found that the seeding culture is a pure culture, finds cinematically and filamentary growth, when what oterom apical (apical) cell consisted entirely of cytoplasm, while the older cells were slightly vacuolation. the pH of the suspension culture was 5-5,2, a PMV was determined by centrifugation of up to 10%-15%. In line with this, the culture was suitable for transformation canrenone 11α-hydroxybenzene.

Canrenone (1 g) finely crushed to a particle size of about 5 microns and suspended in sterile water (20 ml). To this suspension was added a 40% (wt./about.) sterile glucose solution; 16% (wt./about.) sterile solution autorizovanych yeast and sterile antibiotic solution; the content of all the ingredients listed for 0 hours reaction time in Table 5. The antibiotic solution was obtained by dissolving sulfate kanamycin (40 mg), tetracycline·HCl(40 mg) and cephalexin (200 mg) in water (100 ml). A suspension of the steroid, a glucose solution, and the solution autorisierung yeast was gradually added to the culture contained in a shaker flask.

Table 5.
Typical additions of steroid and solutions (additives and antibiotics) in the process of biological transformation canrenone in shaker flask
Reaction time, hoursSuspension steroidGlucose mlAvtorizovanniy yeast ml The antibiotic solution, ml
mlproblem
015010,51
8210021
24210010,51
32525021
48210010,51
56525021
72315010,51
90

By passing the reaction, the reaction mixture was periodically analyzed to determine glucose and using thin-layer chromatography was determined by the degree of transformation in the 11α-hydroxybenzene. During the reaction, to the mixture for reaction fermentation was added an additional amount canrenone substrate in controlled quantities for the holding of the glucose level of the order of about 0.1% of the mass. Scheme add to steroid suspension of glucose solution autorizovanych yeast and antibiotic solution are presented in Table 5. The transformation reaction was carried out for 96 hours at 25°C on a rotary shaker (200 rpm and displacement of 5 cm). During fermentation the pH was between 4.5-6. When PMV rose to 60% or above, 10 ml portion of the broth culture was removed and replaced with 10 ml of distilled water. The disappearance canrenone and the emergence of 11α-hydroxykynurenine was continuously monitored during the reaction by sampling at intervals through 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 completion of the reaction, determined on these samples are shown in Table 6.

/tr>
Table 6.
The time response of biological transformation canrenone in shaker flask
Time clockThe degree of transformation
Canrenone Rf.

RF.=0,81
11α-hydroxybenzene

RF.=0,29
01000,0
45050
72080
232080
313070
472080
553070
712575
801585
96˜10˜90

Example 2.

Primary culture in the seed flask was obtained by the method described in Example 1. Received nutrient mixture having the composition indicated in Table 7

Table 7.
Culture for transformation into a 10 liter glass fermenter
numberg/l
Glucose80 g20
Peptone80 g20
avtorizovanniy yeast80 g20
antifoam SAG 4710.5 g
deionized waterDostal to 4 l
sterilization of empty fermenter for 30 min at 130°
- download 3 liters of deionized water on revenue at 40°
- add while stirring the components of the environment
- stirring for 15 minutes, bringing it to $ 3.9 l
- pH of 5.1
- bringing to 5.8 using 20 wt. -%/about. NaOH,
- sterilization at 120°×20 minutes
pH after sterilization =5.5 to 5.7

Initial load of this nutrient mixture (4 l) was injected into the fermenter for transformation with geometric volume of 10 L. the fermenter had a cylindrical configuration and the ratio of height to diameter, which was 2,58. He was equipped with a turbine stirrer (400 rpm)with two disc wheel # 2 with 6 blades each. The external diameter of the wheels of the mixer was 80 mm, each blade had a radial size of 25 mm and height 300 mm, with the upper wheel was located at a distance of 280 mm below the top of the vessel, the bottom wheel was located at 365 mm below the top of the vessel, and partitions for vessel had a height of 210 mm and located radially in the inner part at a distance of 25 mm from the internal is her vertical wall of the vessel.

Seed culture (40 ml) was mixed with nutrient loading in the fermenter and culture for transformation was obtained by incubation for 22 hours at 28°With, at the speed of aeration of 0.5 l/l/min and at a pressure of 0.5 kg/cm2. After 22 hours PMV culture was 20-25%, and pH=5-5,2.

Received a suspension containing canrenone (80 g) in sterile water (400 ml), and 10 ml portion was added to the mixture in the fermenter for transformation. At the same time added a 40% (wt./about.) sterile glucose solution, 16% (wt./about.) sterile solution autorizovanych yeast and sterile antibiotic solution in the ratios shown in Table 8 on the reaction time of 0 hours. The antibiotic solution was obtained by the method described in Example 1.

Table 8.
Typical additions of steroid and solutions (additives and antibiotics) in the process of biological transformation canrenone a 10-liter glass fermentor
Reaction time, hoursSuspension of the steroidGlucose mlAvtorizovanniy yeast mlThe antibiotic solution, ml
mlapprox. g
1042512,540
42512,5
81042512,5
122512,5
161042512,5
202512,5
241042512,540
281042512,5
3212,552512,5
3612,552512,5
4012,552512,5
4412,552512,5
4812,552512,540
5212,552512,5
5612,552512,5
6012,552512,5
6412,552512,5
6812,552512,5
7212,552512,540
7612,552512,5
80
84
88

By passing the reaction, the reaction mixture was periodically analyzed to determine glucose and using thin-layer chromatography was determined by the degree of transformation in the 11α-hydroxybenzene. On the basis of TLC analysis, the reaction BU is gone, carried out as described below, to the reaction mixture was added an additional amount canrenone as consumption canrenone substrate. Conducted monitoring of glucose levels and, when the glucose concentration fell to about 0.05% wt. or below, was added an additional amount of glucose solution to bring the concentration up to about 0.25% wt. During the reaction cycle at different time intervals was also added nutrients and antibiotics. The scheme of adding a steroid suspension, glucose solution, solution autorizovanych yeast and antibiotic solution are presented in Table 8. The transformation reaction was continued for 90 hours at a speed of aeration 0.5 volume of air per volume of liquid per 1 minute (rpm rpm) at a positive pressure at the head of 0.3 kg/cm2. The temperature was maintained at 28°up until PMV reaches 45%, after which the temperature was lowered to 26°and this temperature was maintained with an increase in the PMV from 45% to 60%, and then it was maintained at 24°C. the Initial rate of stirring was 400 rpm, and after 40 hours was increased to 700 rpm pH maintained in the range of 4.7 and 5.3 by addition of 2M phosphoric acid or 2M NaOH as indicated. As foaming was suppressed by adding a few drops of antifoam SAG 471. During the reaction the AI disappearance canrenone and the emergence of 11α -hydroxykynurenine traced with time intervals of 4 hours, TLC analysis of samples of the broth. When the broth has disappeared largest number canrenone, I added an additional amount canrenone.

After adding the total number canrenone reaction was completed, when TLC analysis showed that the concentration canrenone substrate for 11α-hydroxyquinolone product has dropped to about 5%.

At the completion of the reaction cycle broth for fermentation was filtered through muslin cloth to separate the mycelium from the liquid broth. The fraction of mycelium resuspendable in ethyl acetate with approximately 65 volumes (5.2 liters) per gram canrenone loaded in the reaction. Suspension of mycelium in ethyl acetate was heated under reflux for one hour with stirring, and then cooled to about 20°and was filtered on a Buechner funnel. Mycelial residue was sequentially washed with ethyl acetate (5 vol. one gram canrenone download; 0.4 l) and deionized water (500 ml) to remove an ethyl acetate extract of the sediment. The filter cake is discarded. Enriched with the extracts, wash with solvent and water washing was collected in the separator, and then left to stand for 2 hours for phase separation.

Then the aqueous phase was discarded, and the body of the ical phase was concentrated in vacuo to obtain a residual volume of 350 ml VAT residue was cooled to 15°and this temperature was maintained with stirring for about one hour. The resulting suspension was filtered to remove the crystalline product, and the filter residue was washed with ethyl acetate (40 ml). After drying were determined output 11α-hydroxykynurenine, who was 60,

Example 3.

The spore suspension was obtained from the standard crops in the manner described in Example 1. In a 2000-ml round bottom flask with baffles (3 partitions, each of size 50 mm × 30 mm)containing nutrient solution (500 ml)having the composition indicated in Table 4, was injected an aliquot (0.5 ml) suspension of spores. The resulting mixture was incubated in a flask for 24 hours at 25°on the shaker periodic action (shaking at 120 min; offset 5 cm), resulting in a received culture, which was established after its observation under the microscope, was a pure culture with hyphae, well twisted into a spiral. the pH of the culture was approximately 5.3 to 5.5, a PMV (as determined by centrifugation at 3000 rpm for 5 min) was 8-10%.

Using the thus obtained culture seed culture was obtained in the fermenter stainless steel, having a vertical cylindrical configuration, the geometrical volume of 160 l and relative f the RIAT 2,31 (height =985 mm, diameter =425 mm). The fermenter was equipped with a turbine stirrer (400 rpm)with two disk working wheels, each of which had 6 blades with an outer diameter of 240 mm, and each blade had a radial size of 80 mm and height 50 mm 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. Vertical walls had a height of 890 mm and located radially inside at a distance of 40 mm from the inner vertical wall of the fermenter. The mixer was run at speed 170 rpm First in the fermenter was introduced nutrient mixture (100 l), having the composition indicated in Table 9, and then entered the portion of the pre-inoculum (1 l), obtained as described above and having a pH of 5.7.

Table 9.
For vegetative culture in a 160 liter fermenter, about 8 l, required for seed production fermenter
numberg/l
Glucose2 kg20
Peptone2 kg20
avtorizovanniy yeast2 kg20
antifoam SAG 4710,010 kgmark is the first number
deionized waterDostal up to 100 l
sterilization of empty fermenter for 1 hour at 130°
download 6 l of deionized water, heated at 40°
add, stirring, components of the environment;
- stirring for 15 minutes,
- bringing to volume with 95 l
- sterilization at 121°With 30 minutes
pH after sterilization of 5.7
add sterile deionized water to 100 l

Inoculated mixture is incubated for 22 hours at a speed of aeration of 0.5 l/l/min and at a pressure in the head part of 0.5 kg/cm2. The temperature was maintained at 28°up until PMV not reached 25%, after which the temperature was lowered to 25°C. the pH was maintained in the range of 5.1 and 5.3. The increase in the volume of the mycelium shown in Table 10, which also shows the pH and profiles of dissolved oxygen in the reaction sowing culture.

Table 10.
The growth of the mycelium by the reaction of fermentation seed culture
The period of fermentation, hourspHThe amount of precipitated mycelium (PMV),% (3000 rpm/5 min)Dissolved oxygen, %
05,7±0,1100
45,7±0,1100
85,7±0,112±385±5
125,7±0,115±372±5
165,5±0,125±540±5
205,4±0,130±535±5
225,3±0,133±530±5
245,2±0,135±525±5

Using the thus obtained seed culture fermentation cycle for transformation was carried out in a vertical cylindrical fermenter stainless steel having a diameter of 1.02 m, height 1.5 m and the geometric volume of 1.4 m3. The fermenter was equipped with a turbine mixer having two working wheels, one of which races regalos 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 has six blades, each having a radial size of 95 cm and a height of 75 see Vertical walls had a height 1440 mm and located radially inside the reactor at a distance of 100 cm from the inner vertical wall of the fermenter. Was received nutrient mixture (100 l), having the composition specified in Table 11:

Table 11.
Culture for biological transformation in 1000 l fermenter
numberg/l
Glucose16 kg23
Peptone16 kg23
avtorizovanniy yeast16 kg23
antifoam SAG 4710.080 kgtracking number
deionized waterDostal up to 700 l
sterilization of empty fermenter for 1 hour at 130°
download 600 l of deionized water;
- heated at 40°
- stirring for 15 minutes,
- bringing to volume of 650 l
- sterilization at 121°With 30 minutes
pH after sterilization =5,7
add sterile deionized water up to 700 litres

Bootstrapping (700 l) of this nutrient mixture (pH=5,7) was injected into the fermenter, and then entered the seed inoculum of this example (7 l), obtained as described above.

Nutrient mixture containing the inoculum, incubated for 24 hours at a speed of aeration of 0.5 l/l/min and at a pressure in the head part of 0.5 kg/cm2. The temperature was maintained at 28°and the mixing rate was 110/min, the Increase in the volume of the mycelium are shown in Table 12, which also shows the pH and profiles of dissolved oxygen in the reaction sowing culture.

Table 12.
The growth of the mycelium in the fermenter culture for fermentation
The period of fermentation, hourspH The amount of precipitated mycelium (PMV),% (3000 rpm/5 minutes)Dissolved oxygen, %
05,6±0,2100
45,5±0,2100
85,5±0,212±395±5
1215±390±5
165,4±0,120±575±5
205,3±0,125±560±5
225,2±0,130±540±5

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

To the thus obtained culture for transformation was added to the suspension canrenone (1,250 kg; finely ground to a particle size of 5 microns) in sterile water (5 l). Sterile solution additives and the antibiotic solution was added in the ratios shown in Table 14 on the reaction time of 0. The composition of the solution of the additives shown in Table 13.

Table 13.
Solution additives (for transformative culture)
number
Dextrose40 kg
Avtorizovanniy yeast8 kg
Antifoam SAG 4710,010 kg
deionized waterDostal up to 100 l
- sterilization of empty 150 l fermenter for 1 hour at 130°
download 70 l of deionized water, heated at 40°
add, while stirring, the components of solution additives"
the stirring for 30 minutes, bringing to volume with 95 l
- pH of 4.9
- sterilization at 120° × 20 minutes
pH after sterilization nearly 5

Biological transformation was carried out for about 96 hours at a speed of aeration of 0.5 l/l/min and at a pressure in the head part of 0.5 kg/cm2and pH was 4.7 and 5.3, which is corrected, if necessary, by adding 7,5M 4M NaOH or H3PO4. The initial rate of aeration was 100 rpm, and then after 40 hours was increased to 165 rpm, and 64 hours to 250 rpm mentallychallenged temperature was 28° And then, when PMV reached 45%, the temperature was lowered to 26°and when PMV was increased to 60%, then the temperature was lowered to 24°C. To regulate the foam, if necessary, added a few drops of SAG 471. The levels of glucose in the reaction of fermentation was monitored with time intervals of 4 hours and when the glucose concentration fell below 1 g/l, this party has added an additional amount of sterile solution additives (10 l). The disappearance canrenone and the emergence of 11α-hydroxykynurenine was monitored during the reaction by HPLC. When at least 90% of the original canrenone turned 11α-hydroxybenzene, was added 1,250 kg canrenone. When the added amount was found 90%conversion canrenone, we introduced an additional 1,250 kg canrenone. Using the same criterion was added an additional amount canrenone (portion 1,250 kg) up until the reactor is not entered all loading (20 kg). After adding the total number canrenone reaction was completed, when the concentration of unreacted canrenone substrate relative to the number produced 11α-hydroxykynurenine was 5%. Diagram add canrenone, sterile solution additives and antibiotic solution shown in Table 14.

Table 14.
Add steroid and solutions (additives and antibiotics) in the process of biological transformation canrenone in the fermenter
Reaction time, hoursCanrenone suspensionSterile solution additives, litersThe antibiotic solution, litersvolume ˜ litres
kgadding kg
01,2501,25108700
410
81,2502,510
1210
161,25010
2010
241,2505108800
281,250Ȋ 10
321,25010
361,25010
401,25010
441,25010
481,25012,5108900
521,25010
561,25010
601,25010
641,25010
681,25010
721,250201081050
760
80
84
88
92
Only

When biological transformation was completed, the mycelium was isolated from the broth by centrifugation in a basket centrifuge. As determined by HPLC, the filtrate contained only 2% from the total number of 11α-hydroxykynurenine in broth to collect and, therefore, has been removed. Then the mycelium is suspended in ethyl acetate (1000 l) in the tank for the extraction capacity of 2 m3. This suspension was heated for one hour under stirring and heating under reflux with ethyl acetate, then cooled and centrifuged in a basket centrifuge. Sediment mycelium was washed with ethyl acetate (200 l), and then poured. The extract from the solvent-enriched steroid, left for one hour dlyaudaleniya the aqueous phase. The aqueous phase was extracted with an additional amount of an ethyl acetate solvent (200 l), and then discarded. Combined phase solvent was osvetleni by centrifugation and placed in the hub (geometric volume of 500 l)and then concentrated in vacuum to a residual volume of 100 L. Upon evaporation of the initial load in the hub of the combined extract and wash solutions was 100 l and the volume was maintained constant by continuously or periodically adding a combined solution as the volatility of the solvent. After the stage of evaporation distillation residues were cooled to a temperature of 20°and was stirred for two hours and then filtered on a Buchner filter. The vessel, the hub was washed with ethyl acetate (20 l) and this wash solution is then used to wash the precipitate on the filter. The obtained product was dried in vacuum for 16 hours at 50°C. Output 11α-hydroxykynurenine was 14 kg

Example 4.

Liofilizovannye spores of Aspergillus ochraceus NRRL 405 suspended in the medium for the cultivation liquid corn extract (2 ml)having the composition specified in Table 15 below:

Table 15.
Environment with liquid corn extract (Environment is Multivitamine primary seed culture)
Liquid corn extract30 g
Yeast extract15 g
Monobasic phosphate ammonium3 g
Glucose (download after sterilization),

distilled water is sufficient to 1000 ml pH of 4.6, brought to pH 6.5 by adding 20% NaOH;

the distribution of 50 ml in 250 ml Erlenmeyer flasks;

sterilization at 121°C for 20 minutes.
30 g

The resulting suspension was used to inoculate reproduction of spores on agar plates. Prepare ten cups with agar, each of which contained a solid medium for cultivation: glucose/yeast extract/phosphate/agar having the composition specified in Table 16:

Table 16.
GYPA (glucose/yeast extract/phosphate/agar Cup
Glucose (download after sterilization)10 g
Yeast extract2.5 g
K2HPO43 g
Agar

distilled water, a sufficient quantity to 1000 ml, bringing the pH to 6.5, sterilization at 121°With 30 minutes
20 g

0.2 ml aliquot of the suspension was transferred to apowersoft each Cup. These cups were incubated for ten days at 25°C, after which the spores from all cups were collected in sterile cryogenic protective environment, having the composition specified in Table 17:

Table 17.
ALL/glycerol (glucose/yeast extract/phosphate/agar for vessels with uterine environment)
Glucose (download after sterilization)10 g
Yeast extract2.5 g
K2HPO43 g
Glycerine

distilled water, a sufficient quantity up to 1000 ml sterilize at 121°With 30 minutes
20 g

The resulting suspension was distributed in 20 vessels, with each vessel was transferred to 1 ml of suspension. These vessels contained the basic Foundation of the cells, which can be used for producing the working Fund of the cells in order to generate inoculum for biological transformation canrenone 11α-hydroxybenzene. These vessels containing the primary cell Bank, kept in the freezer in the vapor phase of liquid nitrogen at -130°C.

To start production of the working Fund of the cells of the spores from one vessel with an initial Fund of cells resuspendable in a sterile environment for cultivation (1 ml)having the composition, specified in Table 15. This suspension was divided into ten 0.2 ml aliquot and each aliquot was used for inoculation of agar plates with solid medium for the cultivation, having the composition specified in Table 16. These cups were incubated for ten days at 25°C. On the third day of incubation the lower part of the environment for cultivation had a brown-orange color. At the end of incubation was observed intensive production of spores, with Golden color. Spores from each Cup were collected by the method described above to obtain the source cell Foundation. Just prepare one hundred vessels, each of which contained 1 ml of suspension. These vessels contain a work cell Foundation. Vessels with a working cell Foundation also kept in the freezer in the vapor phase of liquid nitrogen at -130°C.

Medium for cultivation (50 ml)having the composition indicated in Table 15, were loaded into a 250 ml Erlenmeyer flask. Into a flask were introduced aliquot (0.5 ml) of the working cell suspension and mixed with the medium for cultivation. Inoculated mixture is incubated for 24 hours at 25°for producing a primary seed culture having a volume of precipitated mycelium approximately 45%. After visual observation of the culture it was found that it contains granulomatosis mycelium diameter 1 to 2 mm; but after observation under a microscope, it was identified as a pure culture.

The cultivation of secondary seed culture was initiated by introducing a medium for cultivation, having the composition indicated in Table 15, 2.8-liter flask of Fernbach with subsequent inoculation of the medium part (10 ml) primary seed culture of this example, the receipt of which was described above. This inoculated mixture is incubated at 25°within 24 hours on a rotary shaker (200 rpm, shift - 5 cm). After incubation the culture possessed the same properties that were described above for the primary seed culture, and this culture can be used for the reaction of transformation by fermentation, in which canrenone biologically turns 11α-hydroxybenzene.

Transformation was carried out in the patch BIOSTAT fermenter Braun E, description of which is presented below:

Capacity15 litres with a round bottom
Height53 cm
Diameter20 cm
Height/diameter2,65
Impellerthe diameter 7,46 cm, 6 blades 2.2×1,4 cm
The location of the paddle-wheels65,5, 14.5 and 25.5 cm from the bottom of reservoirs is and
Partitionsfour; 1,9×48 cm
Bubblerthe diameter of 10.1 cm, 21 hole
Temperature controlis provided with an outer jacket casing

Canrenone at a concentration of 20 g/l suspended in deionized water (4 l) and part (2 l) environment for cultivation, having the composition indicated in Table 18, was added while stirring the mixture in the fermenter at 300 rpm

Table 18.
Growth medium for biological transformation of culture in 10 l fermenter
numbernumber/l
glucose (download after sterilization)160 g20 g
Peptone160 g20 g
yeast extract160 g20 g
antifoam SAG 4714,0 ml0.5 ml
canrenone

deionized water Dostal to 7.5 l

sterilization of empty fermenter for 30 min at 121°
160 g20 g

The resulting suspension is stirred for 15 minutes, after which the volume was brought to 7.5 l of patentabilty deionized water. At this stage the pH of the suspension was brought to 5.2 to 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 the fermenter E. Braun pH after sterilization was 6.3±0,2 and the final volume was 7.0 liters of Sterilized suspension was inoculable part (0.5 l) secondary seed culture of this example, which was obtained as described above, and the volume brought up to 8.0 l by adding 50% sterile glucose solution. Fermentation was carried out at a temperature of 28°up until PMV did not reach 50%, after which the temperature was lowered to 26°and then, when PMV exceeded 50%, the temperature was lowered to 24°to maintain a constant level PMV below approximately 60%. The air was introduced through a bubbler at a speed of 0.5 rpm rpm based on the initial volume of liquid and the pressure in the fermenter was maintained at around 700 millibar. Stirring was started with a speed of 600 rpm, and then, if necessary, gradually increased to 1000 rpm to maintain dissolved oxygen level above 30%. The glucose concentration was continuously monitored. After initially high glucose concentration fell below 1%, thanks to its acquisitions during the fermentation reaction, was added an additional amount of glucose in the form of 50%sterile glucose solution to support the project for its concentration to within 0.05%-1% throughout the remainder of the cycle for this party. Before inoculation the pH was 6.3±0,2. After lowering the pH to about 5.3 during the initial period of fermentation, the pH was maintained in the range 5.5±0,2 during the remaining cycle by adding ammonium hydroxide. Foaming was regulated by adding polietilenglikoli defoamer, commercially available under the trademark SAG 471 OSI Specialties, Inc.

The culture growth was observed in the first 24 hours cycle, during which the PMV was about 40%, the pH was about 5.6, and dissolved oxygen content was about 50%. Turning canrenone began even during culture growth. Concentration canrenone and 11α-hydroxykynurenine were observed during the biological transformation through a daily analysis of samples. Samples were extracted with hot ethyl acetate and the solution obtained sample was analyzed using TLC and HPLC. Biological transformation was considered complete if residual canrenone was about 10% of the initial concentration. Approximate time of conversion was $ 110-130 hours.

After completion of the biological transformation of mycelial biomass was isolated from the broth by centrifugation. The supernatant was extracted with equal volume of ethyl acetate, and the aqueous layer is discarded. Mycelial fraction resuspendable in ethyl acetate with COI is whether the approximately 65 volume per gram canrenone, loaded into the reactor for fermentation. Then mycelial suspension was heated under reflux for one hour, while stirring, and then cooled to about 20°and was filtered on a Buechner funnel. Mycelial residue on the filter is washed twice with 5 volumes of ethyl acetate per gram canrenone loaded into the fermenter, and then washed with deionized water (1 l) to remove residual ethyl acetate. Aqueous extract enriched solvent washing solvent and water washing were combined. The remaining depleted mycelial residue or rejected, or again were extracted depending on the result of the analysis on presence of residual steroids. The combined liquid phases were left for two hours to defend. And the organic phase was concentrated in vacuum until the residual volume does not become equal to approximately 500 ml. Then the vessel of the apparatus was cooled to about 15°by slow mixing for approximately 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 vacuum at 50°C.

Example 5.

Liofilizovannye spores of Aspergillus ochraceus ATCC 18500 suspended in the culture medium liquid to cook the bowler extract (2 ml), as described in Example 4. Ten agar cups received method, also described in Example 4. These cups were incubated and the spores were collected as described in Example 4 to obtain primary cell Foundation. The vessels containing the main cellular Fund, kept in the freezer in the vapor phase of liquid nitrogen at -130°C.

From the vessel with the main cell Foundation received a work cell Fund, as described in Example 4, and stored in the freezer in liquid nitrogen at -130°C.

Medium for cultivation (300 ml)having the composition specified in Table 19, were loaded into a 2 l flask with baffles. To this flask was injected an aliquot (3 ml) of the working cell suspension. Inoculated mixture is incubated for 20-24 hours at 28°C on a rotating shaker (200 rpm, displacement of 5 cm) with the production of primary seed culture, which had a volume of precipitated mycelium approximately 45%. After visual inspection, it was found that this culture contains granulomatosis mycelium with a diameter of 1-2 mm; and after observing under a microscope, it was found that this culture is pure.

Table 19.
Media for cultivation of primary and secondary seed culture
Glucose (download erased after the implementation) 20 g
Peptone20 g
Yeast extract20 g
distilled water, a sufficient quantity up to 1000 ml sterilize at 121°With 30 minutes

The cultivation of secondary seed culture was initiated by the introduction of 8 l of medium for culturing having the composition specified in Table 19, in 14-liter glass fermenter. This fermenter was inoculable 160-200 milliliters primary seed culture of this example. Obtaining this culture described above.

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

The transformation was performed in 60 l fermenter, mainly by the method described in Example 4, except that the medium for culturing had the composition indicated in table 20, and the initial loading of the secondary seed culture was 350-700 ml stirring Speed first was 200 rpm, but then it was increased to 500 rpm because of the need to maintain the level of dissolved oxygen above 10%. The approximate time of biological transformation to 20 g/l canrenone status is ulala 80-160 hours.

Table 20.
Growth medium for biological transformation of culture in 60 l fermenter
numbernumber/l
glucose (download after sterilization)17,5 g0.5 g
Peptone17,5 g0.5 g
yeast extract17,5 g0.5 g
Canrenone (download in the form of a 20% suspension in sterile water)700 ml20 g
deionized water sufficient to 35 l, sterilization for 30 min at 121°

Example 6.

Using a suspension of spores from the working cell of the Fund produced by the method described in Example 4 were obtained primary and secondary crops, mostly with the method described in Example 4. Using a secondary seed culture produced in this way was carried out by two cycles of the biological transformation in accordance with the modified method, illustrated in figure 1, and was carried out by two cycles of the biological transformation method, illustrated in figure 2. Cultural environment for transformat and, diagram add canrenone, time of collection and the degree of transformation of these cycles are shown in Table 21. In the cycle R2A used the scheme of adding canrenone, mainly described the Example 3, whereas in the cycle R2C used a modified scheme of Example 3, with only two additions canrenone, one at the beginning of the cycle, and another 24 hours. In cycles R2B and R2D all loading canrenone was introduced in the beginning of the cycle and this process was carried out mainly by the method described in Example 4, except that before the introduction of the fermenter download canrenone sterilized in a separate vessel, and glucose was added as the reaction of the party. To minimize the formation of lumps formed after sterilization, used the homogenizer of Moringa. In cycles R2A and R2B canrenone was introduced into the loading party in a solution of methanol and, in this respect, these cycles also differed from the cycles described in Examples 3 and 4, respectively.

In cycles R2A and R2B concentration of methanol in the fermented medium was accumulated to approximately 6.0% and this concentration has been found to inhibit the growth of culture and biological transformation. However, based on the results of these cycles, it was concluded that methanol or other miscible with water, the solvent can be successfully used when Bo is its low concentrations to increase download canrenone and ensuring the availability of canrenone in the form of fine sediment, which leads to the increase in the area of the interfacial surface and contributes to the delivery canrenone in the reaction zone.

Canrenone is stable at a temperature sterilization (121° (C)but forms clumps. For crushing these lumps into fine particles using a homogenizer of Moringa that contributes to the successful transformation canrenone the desired product.

Example 7.

Using a suspension of spores from the working cell of the Fund produced by the method described in Example 4 were obtained primary and secondary crops, mostly with the method described in Example 4. Description and results of Example 7 shown in Table 22. Using a secondary seed culture, producirovanie 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 terms in Example 5. In the last three cycles (R3A, R3B and R3D) canrenone sterilized in a portable tank together with the medium for cultivation that does not contain glucose. Glucose was filed from another tank in the conditions of asepsis. Sterilized suspension canrenone introduced into the fermenter or before inoculation or during the early stages of biological transformation. In the cycle R3B additional quantity sterile is amrinone and medium for cultivation was introduced through 46.5 hours. Lumps canrenone formed after sterilization, were crushed using a homogenizer of Moringa that contributed to the production of finely dispersed suspension is introduced into the fermenter. Transforming the environment for cultivation, the scheme of adding canrenone, schema, add nutrients, time of collection and the degree of transformation for these cycles are presented in Tables 22 and 23.

Thanks to the growth of filaments in the fermenter was observed highly viscous broth in all four cycles in this Example. To remove obstacles, which creates a high viscosity in relation to aeration, mixing, regulation of pH and temperature control during these cycles increased the rate of aeration and the rate of mixing. Reaction conversion was held satisfactorily even under more severe conditions, but on the surface of the liquid broth was formed dense precipitate. Thanks to this draft, some amount of unreacted canrenone was recovered from the broth.

Example 8.

Description and results of Example 8 systematized in Table 24. Were four cycle fermentation, in which 11α-hydroxybenzene was produced by biological transformation canrenone. In two of these cycles (R4A and R4D) biological TA is giving exercised, basically, the same way that was described for cycles R3A and R3D in Example 6. In the cycle R4C canrenone was turned into an 11α-hydroxybenzene, basically, the same way that was described in Example 3. In the cycle R4B process was carried out mainly by the method described in Example 4, i.e. sterilization canrenone and media for culturing in the fermenter immediately before inoculation; all nitrogen and phosphorus nutrients were introduced in the beginning of the cycle, and the additional solution containing only glucose was injected into the fermenter to maintain glucose levels as you progress through the cycle. In the last process (cycle R4B) the concentration of glucose was monitored every 6 hours and the glucose solution was added in a specified amount to maintain glucose levels in the range of 0.5 to 1%. Schema add canrenone these cycles are presented in Table 25.

All fermenters were performed high-speed stirring and aeration during the greater part of the cycle, fermentation, because fermented environment became highly viscous after one day and then after inoculation.

Example 9.

Transforming the environment for cultivation, schemes add canrenone, time of collection and the degree of transformation for cycles in this Example, before the taulani in Table 26.

Four cycles of biological transformation was performed basically as described for cycle R4B in Example 8, except for the procedures described below. In the cycle R5B upper impeller turbine disk mixer used for mixing in other cycles, replaced sea rotor to pump down. The effect of pumping from the top down provides axial flow of the broth in the center of the fermenter and contributes to a reduction in the formation of a dense precipitate. Methanol (200 ml) was added immediately after inoculation loop R5D. Because canrenone in the fermenter was sterilized, all nutrients, except glucose was given in the beginning of the cycle, thereby avoiding the need for supply chain nitrogen sources, phosphorus sources, or antibiotics.

To ensure wetting of the solid phase, acting on the surface of the liquid in each fermenter through 96 hours after the start of the cycle was added to the medium for cultivation (2 l). The problems associated with mixing, can be generally overcome by either adding media for culturing or by using an impeller pump pump down (cycle R5B), but the results of these cycles indicate the feasibility and benefits of this approach and suggest that sufficient razmeshivayut to be carried out in accordance with standard practice.

Example 10.

Three cycles of biological transformation was performed mainly by the method described in Example 9. Transforming the environment for cultivation, schemes add canrenone, time of collection and the degree of conversion cycles for this Example are presented in Table 27.

Environment for the cultivation of (1.3 l) and sterile water (0.8 l) was added over 71 hours per cycle R6A for impregnation of thick sediment mycelium exposed above the surface of liquid broth. For the same purposes medium for cultivation (0.5 l) and sterile water (0.5 l) was added over 95 hours in the cycle R6B. Data on the balance of the materials showed that the mass balance is better to determine in the case when the level of the dense sludge exposed above the liquid surface, is minimized.

Example 11.

Were held cycles fermentation to compare pre-sterilization canrenone with sterilization canrenone and media for culturing in the fermenter for transformation. Cycle R7A carried out by the method illustrated in figure 2, in terms comparable to the terms of cycles R2C, R2D, R3A, R3B, R3D, R4A and R4D. Cycle R7B is illustrated in Figure 3 in terms comparable with the terms of cycles carried out as described in Examples 4, 9 and 10 and cycle R4B. Transforming the environment for cultivation, schemes add canrenone, time of collection and the step is no turning cycles for this Example are presented in Table 28.

Table 28.
A description of how the experiment on the 10 l scale biological transformation
The cycle numberR7AR7B
Medium (g/l)cycle similar to the cycle R7A
liquid corn extract30
yeast extract15
NH4H2PO43
glucose15
OSA0.5 ml
pHbrought to 6.5 by adding 2,5N NaOH
Download canrenone160 g canrenone sterilized and stirred out of the fermenter160 g canrenone sterilized in the fermenter
Boot environmentThe supply of glucose; canrenone added with 1.6 l of medium for cultivationThe supply of glucose; other substances were not added
Collection time118, 5 hours118,5 hour
Biological transformation93%89%

A mass balance on the a certain on the basis of a finite sample, taken from the party R7B, was 83%, which indicated that a significant loss or decomposition of the substrate in biological transformation was not observed. It was found that the mixing for both cycles was adequate.

The residual glucose concentration exceeded the reference range preferably 5-10 g/liter in the first 80 hours of reaction. Loose sediment that has accumulated in the head part of both fermenters, has not had any noticeable adverse effect on the performance of the loop.

Example 12.

The extraction efficiency was determined in a series of cycles 1 l extraction and systematized in Table 29. In each of these cycles, steroids were extracted from mycelium using ethyl acetate (volume fermentation 1 l/l). In each cycle was performed two sequential extraction. Based on RP-HPLC at the first extraction was allocated approximately 80% of the steroid; and when the second extraction, the output of the steroid was increased to 95%. In the third extraction was to be allocated another 3% of the steroid. The remaining 2% of the steroid was loss in the aqueous phase of the supernatant liquid. The extract was evaporated to dryness in a vacuum, but flushing any additional solvent is not performed. Processing solvent should contribute to the improvement of the output of the initial extraction from the point of view of economic costs.

Table 29.Selection 11α-hydroxykynurenine in 1-liter of extraction (%)The cycle number1st extract2nd extract3rd extractThe supernatantR5A79%16%2%2%R5A84%12%2%2%R4A72%20%4%4%R4A79%14%2%5%R4B76%19%4%1%R4B79%16%3%2%R4B82%15%2%1%Average79%16%3%2%

Methyl isobutyl ketone (MIBK) and toluene were evaluated as solvent extraction/crystallization for 11α-hydroxykynurenine on a scale of 1 l of broth. Using the above schema extraction with MIBK and toluene were comparable with ethyl acetate as extraction efficiency, and productivity of crystallization.

Example 13.

In cachestatistics assessment methods, illustrated in figure 2 and 3, the study of particle size was performed using canrenone substrate added at the beginning of the fermentation cycle in each of these ways. As described above, in the method, illustrated in figure 1, canrenone before its introduction into the fermenter finely crushed, but not sterilized, the growth of undesirable microorganisms inhibited by the addition of antibiotics. In ways, illustrated in figure 2 and 3, canrenone sterilized prior to the reaction. In the method, illustrated in figure 2, canrenone before its introduction in the fermenter was sterilized in the mixer. In the method, illustrated in Figure 3, the suspension canrenone in the medium for the cultivation sterilized in the fermenter at the beginning of the cycle. As discussed above, the sterilization may lead to agglomeration of particles canrenone. Due to the limited solubility of canrenone in the aquatic environment for the cultivation efficiency of the method depends on the mass transfer from the solid phase, and therefore it can be expected that it depends on the area of the interfacial surface defined by the surface area of the particulate solid substrate, which, in turn, depends on the particle size distribution of these particles. These considerations serve primarily as a deterrent in the way illustrated in figure 2 and 3

However, it was found that the mixing in the mixer, Figure 2, and in the fermenter tank, Figure 3, together with the action of the shear pump is used to feed the party, Figure 2, contributes to the destruction of these agglomerates into particles with a size sufficient to enable submission of unsterilized and finely chopped canrenone in the cycle shown in figure 1. This was illustrated by evaluating the particle size distribution for particles canrenone present at the beginning of the reaction cycle of each of the three methods. Cm. Table 30 and Figure 4 and 5.

Table 30.
The distribution of particles for three different samples canrenone
Sample45-125 μm<180 μmthe average size, microns# party: % biological transmutations
Download canrenone75%95%-R3C:

93.1% of (120 h)

R4C:

96,3% (120 h)
Mixed sample31,2%77,2%139,5R3A:

94,6% (120 h)

R4B:

95,2% (120 h)
Sterilized sample24,7%65,1%157,4R4B:

97,6% (120 h)

R5B:

93,8% (120 h)

Based on data from Table 30, it should be noted that the mixers and pump with shear action is effective to decrease the average particle size sterilized canrenone to the same order of magnitude, which is typical for non-sterilized substrate, however, still remains a significant difference in particle size, which argues in favor of unsterilized substrate. Despite this difference, the productivity of the reaction indicate that the method with pre-sterilization is at least as productive as the method illustrated in figure 1. Additional benefits can be realized in the manner illustrated in figure 2, using several stages to further reduce and control the particle size, for example, by wet grinding sterilized canrenone and/or by pasteurization instead of sterilizacii.

Example 14.

Seed culture was obtained by the method described in Example 5. After 20 hours the mycelium in the fermenter with the inoculum had a soft texture with 40% of PMV. Its pH was 5,4, and 14.8 g/l glucose remained neotrezannymi.

Was obtained transforming the environment for cultivation (35 l), having the composition specified in Table 20. To obtain the nutrient medium glucose and yeast EXT the act were sterilized separately and mixed to obtain a homogeneous nutrient mass with an initial concentration of 30 wt%. glucose and 10% of the mass. yeast extract. the pH of the mass was brought to 5.7.

Using this medium (table 20) were conducted two cycles of biological transformation canrenone 11α-hydroxybenzene. Each of these cycles were performed in 60-liter fermenter equipped with a mixer containing one turbine impeller Rushton and two turbine working wheels Lightnin A.

The initial boot media for culturing in the fermenter was 35 HP Finely ground and unsterilized canrenone was added to the initial concentration of 0.5%. The medium in the fermenter was inoculable seed culture obtained as described in Example 5, when the initial degree of inoculation of 2.5%. Fermentation was carried out at a temperature of 28°if the stirring speed is 200-500 rpm, at a speed of aeration of 0.5 rpm rpm and at a pressure sufficient to maintain the dissolved oxygen level of at least 20% vol. Culture for transformation, developing during the production cycle, was in the form of very fine oval granules (about 1-2 mm). Canrenone and additional nutrients were injected into the fermenter through the supply chain, mainly in the manner described in Example 1. Nutritional supplements were administered every four hours in a ratio of 3.4 g of glucose and 0.6 g yeast extract per litre of broth in the fermenter.

In Alice 31 shows the rate of aeration, the stirring speed, the level of dissolved oxygen, PMV and pH, defined in the corresponding time intervals during each of the cycles described in this example and the method of adding glucose during this cycle. Table 32 shows the profile transformation canrenone. Cycle R11A ended after 46 hours; the cycle R11B continued for 96 hours. In the last cycle for 81 hours was achieved 93%conversion; after 84 hours spent another nutritional Supplement mixture, after which the supply of nutrients was completed. It should be noted that a significant change in viscosity occurred in the time interval between supply of nutrients and the completion of a cycle.

Example 15.

Different cultures tested for the efficiency of the biological transformation of canrenone 11α-hydroxybenzene way, mainly described above.

Work cell Fund 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. Medium for cultivation (50 ml)having the composition indicated in Table 18, was inoculable spore suspension (1 ml) from the working cell of the Fund and placed in the incubator. Posin the Yu culture received in the incubator by fermentation for 20 hours at 26° C. the Contents of the incubator was stirred at 200 rpm

Aliquots (2 ml) seed culture of each organism was used for inoculation of flasks for transformation containing medium for cultivation (30 ml), specified in Table 18. Each culture was used for inoculation of the two flasks, and only used six tubes. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36°and 0.5 ml aliquots of this solution were injected into each of the flasks. Biological transformation was carried out mainly under the conditions described in Example 5, with daily addition of 50 wt%. glucose solution (1 ml). Through the first 72 hours spent observing the growth of the mycelium in the respective fermentation flasks for transformation:

ADS 11394 - good uniform growth;

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

ADS 6227b - good growth; mycelial mass is aggregated in clumps.

Samples of broth were analyzed for the degree of biological transformation. After three days fermentation using ATSS 11394 resulted in 80-90%final transformation into the 11α-hydroxybenzene; ATS 11145 gave a conversion of 50%; and ATSS 6227b gave a conversion of from 80 to 90%.

Example 16.

Using the method basically described in Example 15, on the efficiency of Pribram is of canrenone 11α -hydroxybenzene have been tested by other microorganisms. Tested microorganisms and the results of the tests are presented in Table 33.

Example 17.

Various microorganisms were tested for effectiveness in the conversion of canrenone 9α-hydroxybenzene. Sbrasivaya environment, which were obtained for carrying out fermentation cycles of this Example are presented in Table 34.

Table 34.
Soy flour:
Dextrose20 g
soy flour5 g
NaCl5 g
yeast extract5 g
KN2PO45 g
Waterto 1 l
pH7,0
Peptone/yeast extract/glucose:
Glucose40 g
Bactopeptone10 g
Yeast extract5 g
Waterto 1 l
Wednesday Miller-Hinton:
Meat broth300 g
Casinocity17,5 g/td>
Starch1.5 g
Waterto 1 l

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

The original culture was inoculable frozen stocks dispute (20 ml soy flour 250 ml Erlenmeyer flask). Flasks were covered with a filter made of frosted glass and bio-protective coating. The original culture (24 - or 48-hourly) was used for inoculation of cultures metabolism (20 ml in 250 ml Erlenmeyer flask) with 10%-15% of the intersecting volume, then incubated for 24-48 hours and added steroid substrate for the reaction of transformation.

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

The biological transformation products were collected after 5 and 48 hours, or 24 hours after addition of the substrate. The collection was started by adding ethyl acetate (23 ml) or methylene chloride in the fermentation flask. Then number the s was shaken for two minutes and the contents of each flask were poured into a 50 ml conical tube. For separation of the phases, these tubes were centrifuged at 4000 rpm for 20 minutes at room temperature. The organic layer from each tube was transferred into a 20 ml vessel made of borosilicate glass and evaporated in a speed vacuum evaporator. The vessels were covered with a lid and kept at -20°C.

For material in order to determine patterns, scale biological transformations was increased to 500 ml by increasing the number of shaker flasks for fermentation to 25. At collection time (24 or 48 hours after addition of the substrate) into each flask separately was added ethyl acetate, and the flask was covered with a lid and put back in 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 separating funnel, in which the phases are separated under the action of gravity. The organic phase was dried and obtained the crude extract of the steroids contained in the reaction mixture.

The reaction product was analyzed first using thin-layer chromatography on fluorescent facing plates with silica gel (254 microns). To each vessel containing the drained an ethyl acetate extract of the reaction mixture were added ethyl acetate (500 ml). Subsequent analysis was performed using high-performance liquid chromatography and mass spectrometry. T the X-plates showed in a mixture of solvents, chloroform/methanol, 95:5 vol/vol.

Another analysis was performed using high-performance liquid chromatography and mass spectrometry. Used HPLC Waters, software Millennium, the detector photodiode matrix and autonomic samples. In reversed-phase HPLC was used column Waters NovaPak C-18 (particle size 4 μm) with 4 mm cartridge Radial-Pak. Chromatography was started with a linear gradient of solvent (25 minutes) on the speaker initialized in water:acetonitrile (75:25), and finished in water:acetonitrile (25:75). Then used a gradient to 100% acetonitrile (3 minutes) and 4-minute socratous washing, after which the column was regenerated at initial conditions.

For LC/MS ammonium acetate was added as in acetonitrile, and in the aqueous phase at a concentration of 2 nm. In chromatography, it had no significant influence. The eluate from the column was split 22:1, while most of the material was sent to the PDA detector. The remaining 4.5% of material went into the chamber of the mass spectrometer (Sciex API III for ionization electroepilation. Mass spectrometry was carried out in positive mode. A similar series of data obtained by HPLC-chromatogram on the same wavelength with PDA detector, was transferred into a mass spectrometer for the joint analysis of UV and MS data.

Samples mass spectrometry the fragm the procedures can be used for selection of hydroxylated substrates. Two alleged hydroxylated canrenone, 11α-hydroxy - and 9α-hydroxy-lose water at various frequencies and to the extent appropriate, that can be used as diagnostics. In addition, 9α-hydroxybenzene formed ammonium adduct more easily than 11α-hydroxybenzene. In Table 35 systematic data TLC, HPLC/UV and LC/MS for fermentation canrenone and shows which of the tested microorganisms are effective for the biological transformation of canrenone 9α-hydroxybenzene. Of them preferred microorganism is Corynespora cassiicola ADS 16718.

Example 18.

Different cultures tested for the efficiency of the biological transformation of canrenone 11α-hydroxybenzene the method described in General terms above.

Work cell Foundation 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 roseum ATCC 12519 and ATCC 8685 received by way of, basically described in Example 4. Medium for cultivation (50 ml)having the composition indicated in Table 18, was inoculable spore suspension (1 ml) from the desktop to tochnogo Fund and placed in the incubator. Seed culture was obtained in the incubator by fermentation for about 20 hours at 26°C. the Contents of the incubator was stirred at 200 rpm

Aliquots (2 ml) seed culture of each organism was used for inoculation of flasks for transformation containing medium for cultivation (30 ml)listed in Table 15. Each culture was used for inoculation of the two flasks from all 16 tubes. Androstenedione (300 mg) was dissolved in methanol (6 ml) at 36°and 0.5 ml aliquots of this solution were injected into each of the flasks. Biological transformation was carried out mainly in the conditions described in Example 6, within 48 hours. After 48 hours samples of broth were combined and extracted with ethyl acetate as described in Example 17. The ethyl acetate was concentrated by evaporation and the samples were analyzed using thin-layer chromatography to determine whether the product chromatographic mobility, similar to the mobility 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 11α-hydroxyandrostenedione
CultureATC # EnvironmentThe TLC results
Rhizopus oryzae11145CSL+
Rhizopus stolonifer6227bCSL+
Aspergillus nidulans11267CSL+
Aspergillus niger11394CSL+
Aspergillus ochraceusNRRL 405CSL+
Aspergillus ochraceus18500CSL+
Trichothecium roseum12519CSL+
Trichothecium roseum8685CSL+

The data in Table 36 indicate that each of these cultures are able to produce a connection from Androstenedione, which has the same Rf value as the standard 11α-hydroxyandrostenedione.

Aspergillus ochraceus NRRL 405 (ATCC 18500) again tested in the manner described above, and cultural products were isolated and purified using column chromatography on normal phase silica gel using methanol as solvent. Fractions were analyzed by thin layer chromatography. The TLC plate was a plate with Whatman K6F silica gel 60size 10×20, a thickness of 250 is iron. Used solvent mixture chloroform:methanol 95:5 vol/vol. Crystallized product and standard 11α-hydroxyandrostenedione analyzed using LC-MS and NMR spectroscopy. Both compounds had similar profiles and molecular weight.

Example 19A.

Various microorganisms were tested for effectiveness of the biological conversion of Androstenedione 11α-hydroxyandrostenedione way, mainly described above in Examples 17 and 18.

The culture of each microorganism Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC 16888 and ATSS 26693, Epicoccum oryzae ATCC 7156, Curvularia lunata ATCC 12017, Cunninghamella blakesleeana ATCC 8688a and Pithomyces atroolivaceus IFO 6651 were grown by the method basically described in Example 17. Media for culturing and fermentation (30 ml) had the composition specified in Table 34.

11α-hydroxylation of androstendione the above microorganisms were analyzed using mainly methods similar to the methods of identification of the products described in Examples 17 and 18. The results are presented in table 19A-1.

Table 19A-1.
11β-hydroxylation of Androstenedione by various microorganisms
BodyTCXLC/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 is the expected value when the thin-layer chromatography and approximate molecular mass LC/MS.

These results suggest that these microorganisms capable of 11β-hydroxylation of androstendione.

Example 19C.

Various microorganisms were tested for effectiveness of biological transformation maxidone 11β-hydroxybenzene. Sbrasivaya environment for this example was obtained as described in Table 34.

The conditions of fermentation and analytical methods were similar to the methods described in Example 17. The TLC plates and the solvent system described in Example 18. The principle of chromatographic analysis was as follows: 11α-hydroxybenzene and 11α-hydroxybenzene had the same chromatographic mobility, 11α-hydroxybenzene and 9α-hydroxybenzene had the same character mobility, and 111 -hydroxyandrostenedione and 11β-hydroxyandrostenedione. Therefore, 11β-hydroxybenzene must have a mobility similar mobility 9α-hydroxykynurenine. Therefore, the compounds extracted from the culture medium, were tested in comparison with a standard 9α-hydroxykynurenine. The results are presented in Table 37.

Table 37.
Systematic TLC data for education 11β-hydroxykynurenine of maxidone
MicroogranismEnvironment1Nature spot2
Absidia coerula ATCC 6647M,Sbright
Aspergillus niger ATCC 16888S,Rweak (S) ? (R)
Beauveria bassiana ATCC 7159Pbright
Beauveria bassiana ATCC 13144S,P?, ?
Botryosphaeria obtusa IMI 038560weak
Cunninghamella
blakesleeana ATCC 8688aS,Pbright
echinulata ATCC 3655S,Pbright
elegans ATCC 9245S,Pbright
Curvularia lunata ATCC 12017 Sbright
Gongronella butleri ATCC 22822S,Pbright
Penicillium patulum ATCC 24550S,Pbright
Penicillium purpurogenum ATCC 46581S,Pbright
Pithomyces atro-olivaceus IFO 6651S,Pweak
Rhodococcus equi ATCC 14887Mweak
Saccharopolyspora erythaea ATCC 11635M,SFweak
Streptomyces hygroscopicus ATCC 27438M,SFbright
Streptomyces purpurascens ATCC 25489M,SFweak
Thamnidium elegans ATCC 18191S,Pweak
Thamnostylum piriforme ATCC 8992S,Pweak
Trichothecium roseum ATCC 12543P,Sweak (R) ? (S)
1M = Medium Miller-Hinton

P = PYG (peptone/yeast extract/glucose)

S = soy flour

SF = soy flour + formate

2? = disputed contrast control without substrate

These data presumably indicate that the main part of the microorganisms listed in this table, produces a product that is similar or identical 11α-hydroxyechinenone formed from maxidone.

Example 19 (C).

Different mi is reorganise were tested for effectiveness in the conversion of maxidone 11α -hydroxylysine, Δ1,2-maxrenn, 6β-hydroxylysine, 12β-hydroxybenzene and 9α-hydroxybenzene. Maxrenn can be obtained by the method described Weier in U.S. Patent No. 3787396 and R.M.Weier et al., J.Med.Chem., Vol.18, pp.817-821 (1975), which are introduced in the present description by reference. Environment for fermentation were obtained as described in Example 17, except that they was included maxrenn. The fermentation conditions were, in General, similar to the conditions described in Example 17; analytical methods were 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.

Tested microorganisms and the results obtained on their basis, are presented in Table 19 (C)-1.

Table 19S-1.
The production of 11α-hydroxyechinenone of maxidone different microorganisms
The microorganismTCXHPLCm/z 417:399
Beauveria bassiana was ATSS 7159++5:1
Beauveria bassiana was ATSS 13144++10:1
Mortierella isabella was ATSS 42613++1:1
Cunnighamella blakesleeana ADS a ++1:1
Cunninghamella echinulata ADS 3655++1:2
Cunninghamella elegans was ATSS 9245++1:1
Absidia coerula ADS 6647++1:1
Aspergillus niger was ATSS 16888++4:1
Gongronella butieri ADS 22822++3:1
Pithomyces atro-olivaceus was ATSS 6651++3:1
Streptomyces hydroscopicus ADS 27438++3:1

In Table 19S-1 "+" indicates a positive result, that is, Rfis the expected value when the thin-layer chromatography and around the expected retention time in HPLC, m/z 417:399 indicates the ratio of the height of the peaks molecules 417 (hydroxybenzene) and molecules 399 (maxrenn). The standard is the ratio of the height of the peaks of 10:1 for m/z 417 - m/z 399.

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

Table 19S-2.
Production Δ1,2-maxidone of maxidone different microorganisms
The microorganismm/z 399HPLCTCX
Rhodococcus equi ATCC 148875+++
Bacterium cyclo-oxydans ATCC 12673+++
Comomonas testosteroni ATCC 11996+++
Nocardia aurentia ATCC 12674+++
Rhodococcus equi ATCC 21329+++

In Table 19 (C)-2 "+" indicates a positive result, that is, Rfis the expected value when the thin-layer chromatography and around the expected retention time in HPLC, etc.

The product obtained from the Bacterium cyclooxydans ATCC 12673, was isolated from the incubation mixture and analyzed by NMR, which showed that the structural profile of this product meets Δ1,2-macronano. Similarly, the products obtained from other microorganisms listed in Table 19S-2, also believed to be Δ1,2-maxrandom.

Production of 6β and 12β-GI is ratsimandrava

Mortierella isabella ATCC 42613 were grown as described in Example 17, in the presence of maxidone. The fermentation products were isolated and purified using flash chromatography. Purified products were analyzed using LC/MS, as described in Examples 17 and 18, and by using proton NMR and13C-NMR. The data from these tests showed that these products included 6βand 12β-hydroxybenzene.

Table 19S-3.
Production of 9α-hydroxyechinenone of maxidone different microorganisms
The microorganismm/z 417HPLCTCX
Streptomyces hydroscopicus ADS 27438+++
Gongronella butieri ADS 22822+++
Cunninghamella blakesleeana ADS 8688a+++
Cunninghamella echinulata ADS 3655+++
Cunninghamella elegans was ATSS 9245+++
Mortierella isabellina ATCC 42613+++
Absidia coerula ADS 6647+++
Beauveria bassiana was ATSS 7159++ +
Beauveria bassiana was ATSS 13144+++
Aspergillus niger was ATSS 16888+++

The microorganisms listed in Table 19S-3, were grown in the same conditions that were described in Example 17, in the presence of maxidone. The fermentation products were analyzed by TLC and LC/MS as described in Examples 17 and 18. In Table 19S-3 "+" indicates a positive result, that is, Rfis the expected value when the thin-layer chromatography and around the expected retention time in HPLC, etc. These data suggest that the obtained products include 9α-hydroxybenzene.

Example 19D.

Various microorganisms were tested for effectiveness in the conversion of canrenone in Δ9,11-canrenone. Environment for fermentation and culturing conditions were basically the same as in Example 17, except that in this environment was included canrenone. Methods of analysis were similar to the methods described in Example 17 and 18. Microorganisms and the results are presented below in Table 19D-4.

td align="center"> The microorganism
Table 19D-4.
Production Δ9,11-canrenone of canrenone different microorganisms
m/z 339HPLCTCX
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+++

The fermentation products were analyzed by TLC and LC/MS as described in Examples 17 and 18. The symbol "+" indicates a positive result, that is, Rfis the expected value when the thin-layer chromatography and around the expected retention time in HPLC, etc.

The product obtained from Comomonas testosteroni ATCC 11996 was isolated from the culture medium and analyzed using UV spectroscopy. Spectroscopic profile confirmed the presence of Δ9,11-canrenone. Similarly, the products derived from microorganisms listed above in Table 19D-1, presumably also was Δ9,11-canrenone.

Example 20A.

Scheme 1: step 1: Method a: Getting 5'R(5'α),7'β-20'-aminohexanoate-11'β-hydroxy-10 a,13'; -dimethyl-3',5-di oxaspiro[furan-2(3H),17'α(5 N)-[7,4]metheno[4H[cyclopent-[a]phenanthrene]-5'-carbonitrile

In enameled reactor with a capacity of 50 gallons (189,25 DM3) loaded with stirring 61,2 l (of 57.8 kg) DMF, and then 23,5 kg 11-hydroxykynurenine 1. To this mixture was added 7,1 kg of lithium chloride. The mixture was stirred for 20 minutes and loaded 16,9 kg cyanohydrin acetone, and then 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 was added 353 liters of water, and then 5.6 kg of sodium bicarbonate. The mixture was cooled to 0°With, tolerated in enameled reactor capacity 200 gallons (757 DM3and slowly extinguished using 130 kg 6,7% solution of sodium hypochlorite. The product was filtered and washed 3×40 l portions of water with obtaining 21,4 kg raminosoa product.

N1NMR (DMSO-d6): 7,6(2H, sird), a 4.53(1H, d, J=5,9), 3,71(1H, m), 3.0 a to 1.3(17H, m), 1,20(5H, m)0,86(3H, s)0,51 (1H, t, J=10).

Example 20B. Getting 7α-cyano-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-21-carboxylic acid, γ-lactone

50.0 g 11-hydroxykynurenine and of 150.0 ml of dimethylacetamide was added in a clean, dry three-neck flask equipped with a mechanical stirrer, a refrigerator, a thermocouple and a casing for heating. To this mixture, d is balali of 16.0 ml of sulfuric acid solution (obtained by mixing of 50.0 ml of sulfuric acid (98,7% acid grade Baker) to 50.0 ml of water). Then was added a solution of sodium cyanide containing 15.6 g of sodium cyanide and to 27.0 ml of water.

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

Separated methylenchloride layer was washed with 100 ml water and the resulting biphasic mixture was stirred. The mixture is then left for phase separation and separated methylenchloride layer was filtered through 200 g of silica gel (sieve 200-400 mesh, 60, Aldrich). The filtrate was concentrated to dryness under reduced pressure and at a 45°using the device for pumping water, and was about 53,9 g of crude solid product. Then, this crude solid product was dissolved in 50 ml of methylene chloride and was treated with 40 ml of 4n hydrochloric acid in a separating funnel and two-phase mixtures is ü left to defend. Methylenchloride layer was washed with 50 ml water. The combined aqueous layers were extracted with 50 ml of methylene chloride. Then the combined methylenchloride layers were dried with sodium sulfate and received 45 g of solid, which was a mixture of 11α-hydroxykynurenine and product 7α-cyano-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-21-carboxylic acid, γ-lactone.

A sample of this product was analyzed by HPLC (column: 25 cm × 4.6 mm, 5 µm C18'LL Altima); gradient solvent: solvent A=water/triperoxonane acid=to 99.9/0.1, solvent B=acetonitrile/triperoxonane acid=99, 9/0,1, flow rate=1,00 ml/min, gradient=65:30 (about./about.) (As In first); 35:65 (vol./about.) (In 20 minutes); 10:90 (vol./about.) (In 25 minutes), the detector diode matrix, which was found λmax238 nm.

The reaction mixture was analyzed by HPLC-NMR using the following conditions: HPLC column: Bond RX-C8 (25 cm × 4.6 mm, 5 microns) with a gradient solvent of 75% D2O, 25% acetonitrile - 25% D2O, 75% acetonitrile over 25 minutes, with a flow rate of 1 ml/min;

1H NMR spectrum (obtained using suppression of solvent WET) of 5.84 (s, 1H), 4,01(m, 1H), 3,2(m, 1H), 2,9-1,4 (m, a full signal has no significant value due to the suppression of acetonitrile), 0,93 is 0.86(s, overlapping 3H, t,2H).

Note the R 20S. Getting 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) suspended from 46.8 g (to 0.72 mol) of potassium cyanide, 78,6 ml (1,356 mol) of acetic acid and 600 ml of methanol in a three-liter three-neck round-bottom flask. To this mixture was added to 64.8 ml (0.78 mol) of pyrrolidine combined suspension was heated under reflux (64° (C) and held for about 1.5 hours. Then the temperature of the suspension was reduced to 25°S-30°within ten minutes using a cooling bath. 120 ml of concentrated hydrochloric acid was slowly added while cooling, during which he observed the precipitation of solids reddish-brown color.

This mixture was stirred at 25°S-30°C for 1.5 hours, and then after 30 minutes, was added 500 ml of water. This mixture was cooled to 5°in the bath with ice and the pH was brought from 3 to 5.5 (the pH was monitored using strips of indicator paper) by adding 100 ml of water 9,5M sodium hydroxide (0.95 mol). Excess cyanide was dissolved by adding household bleach. 25 ml (at 0.020 mol) was added to conduct a negative test with iodide of starch. The cold mixture (10° (C) was filtered and the solid is s matter was washed with water until while rinsing did not acquire a neutral pH (pH paper). The solid was dried at 60°C to a constant mass byr111.4,

Selected solid melted at 244-246°block Fisher Jones. A methanol solution containing solid substance was not detected absorption in the UV region 210 to 240 nm. IR (CHCl3) cm-12222 (cyanide), 1775 (lactone), 1732 (3-keto).1H-NMR (pyridine-d5) ppm of 0.94(s, 3H), of 1.23(s, 3H).

Example 21A.

Scheme 1: step 2: Getting 4'S(4'α),7'α-hexadeca-hydro-11'α-hydroxy-10'β,13'β-dimethyl-3',5,20'-dioxaspiro-[furan-2(3H),17'β-[4,7]methane[17H]cyclopent-[a]phenanthrene]-5'β(2 N)-carbonitrile

In enameled reactor capacity 200 gallons (757 DM3) were loaded with 50 kg of enamine 2, approximately 445 l 0,8h diluted hydrochloric acid and 75 l of methanol. The mixture was heated for 5 hours to 80°S, and then cooled for 2 hours to 0°C. the Solid product was filtered to obtain 36,5 kg dry diketonato product.

1H NMR (DMSO-d6): a 4.53(1H, d, J=6), 3,74(2H, m), 2,73 (1H, DD, J=14,7), 2,65-2,14 (8H, m), is 2.05(1H, t, J=11), 1,98-1,71(4H, m)of 1.64(1H, m)of 1.55(1H, DD, J=13,5), 1,45-1,20 (7H, m)0,86(3H, s).

Example 21B.

Scheme 1: step 1 and 2: in situ-getting 4'S(4'α),7'α-hexadecagon-11'α-hydroxy-10'β,13'β-dimethyl-3',5,20'-three-oxaspiro[furan-2(3H),17'β(4,7)-meta is about[17H]cyclopent[a]phenanthrene]-5'β (2 N)-carbonitrile of 11α-hydroxykynurenine

In a reactor equipped with a refrigerator, a mechanical stirrer, a casing for heating, automatic regulator and funnel, was loaded with 100 g (280,54 mol) 11-hydroxykynurenine obtained by the method described in Example 1, and then added 300 ml of dimethylacetamide (Aldrich). The mixture was stirred until until 11-hydroxykynurenine was not dissolved. To this mixture was added to 31.5 ml of 50% sulfuric acid (Fisher), which contributed to the increase in the temperature of the mixture to about 10°-15°C. Then a mixture of 11α-hydroxykynurenine within 2-3 minutes was added a solution of sodium cyanide obtained by dissolving 31,18 g (617,20 mol)(Aldrich) of sodium cyanide in 54 ml of deionized water. After adding a solution of sodium cyanide temperature of the mixture was increased to about 20°25°C.

This mixture was heated up to 80°C and maintained at this temperature for 2-3 hours. After HPLC analysis indicated that the reaction conversion of 11α-hydroxykynurenine in the enamine was mostly completed (conversion of more than 98%), the heating source was removed. Without allocation of enamine, present in the mixture, this mixture for 3-5 minutes was added 148 ml of 50% sulfuric acid. Then to the mixture for 10 minutes was added 497 ml of deionized water.

This mixture was heated to 102°is maintained at this temperature until while approximately 500 g of distillate was not removed from the mixture. During the reaction/distillation to the mixture was added 500 ml of deionized water four separate portions 125 ml Each portion was added to the mixture after removal of an equivalent amount of distilled water (approximately 125 ml). The reaction continued for 2 hours. After HPLC analysis indicated that the reaction of hydrolysis of the enamine in the diketone was mostly completed (conversion of 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 three times washed approximately equal portions (about 0.4 l) wash water. In the reactor received 1 l of a solution of methanol and deionized water (1:1, vol/about.) and the filtrate is washed with 500 ml of this solution. Then the filtrate twice washed the remaining 500 ml methanol/water. In the vortex created the vacuum for drying the filtrate to the extent sufficient for it to transfer. The filtrate was transferred into a drying oven where it was dried in vacuum for 16 hours to obtain 84 g of dry diketonato product, 4'S(4'α),7'α-hexadecagon-11'α-hydroxy-10'β,13'β- dimethyl-3',5,20'-dioxaspiro[furan-2(3H),17'β(4,7)-methane-[17H]cyclopent[and]phenanthrene]-5'&x003B2; (2 N)-carbonitrile. HPLC analysis showed 94% of the desired diketone.

Example 22.

Scheme 1: stage 3A: Method a: Getting Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

4-Necked, 5-liter round-bottom flask was equipped with mechanical stirrer, addition funnel to equalize the pressure tube for nitrogen inlet, thermometer and a fridge with a bubbler. This bubbler was connected through a system Tihonovich tubes two 2-liter traps, the first of which was empty and was placed in the reaction vessel to prevent back suction of the material in the second trap (1 l concentrated solution of hydrochloride sodium). In the flask, in 3 l of methanol was added 3-diketone (79,50 g; [weight was not adjusted for purity, which accounted for 85%]). 25% Methanol solution of sodium methoxide (64,83 g) was poured into the funnel and was added dropwise, with stirring in a nitrogen atmosphere for 10 minutes. When you are finished adding orange-yellow reaction mixture was heated under reflux for 20 hours. After this period through an addition funnel to even boiling the reaction mixture was added dropwise 167 ml of 4n HCl (Note: at this stage there is a selection of HCN!). The reaction mixture, which had a light color, becoming pale Golden-orange CEE is and. Then the fridge was replaced head cap for drainage and 1.5 l of methanol was removed by distillation, while the flask through the funnel at the same time was added 1.5 liters of water in accordance with the speed of distillation. The reaction mixture was cooled to room temperature and twice was extracted with 2.25 l aliquot of methylene chloride. The combined extracts are then washed with 750 ml aliquot of cold saturated solution of NaCl, 1N NaOH, and again with saturated NaCl solution. The organic layer was dried with sodium sulfate overnight, filtered and concentrated in vacuo to a volume of ˜250 ml was Then added toluene (300 ml) and the remaining methylene chloride evaporated under reduced pressure, and during this time on the walls of the flask began to form the product as a white solid. The contents of the flask were cooled overnight and the solid was removed by filtration. This substance was washed with 250 ml of toluene, and then twice with 250 ml aliquot of simple ether and dried in vacuum advance-funnel, resulting in a received 58,49 g of a white solid substance, which, as shown by HPLC had a purity of 97.3%. After concentrating the mother liquor was given 6,76 g of product with a purity of 77.1 percent. The total yield, adjusted for purity, was 78%.

1H-NMR (CDCl3): 5,70(1H, s)4,08(1H, s)to 3.67(3H, s), 2,9-1,6(N, m), 1,5-1,2(5H,m)of 1.03(3H, s)./p>

Example 23.

Scheme 1: stage 3B: the Transformation of Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone in Metelitza-17α-hydroxy-11α-(methylsulphonyl)oxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone

5-liter chetyrehkolka the flask was equipped as described above, except that the bubbler was not connected, the system traps. Into the flask under stirring in nitrogen atmosphere was added a complex hydroxyether in the number 138,70 g, and then 1425 ml of methylene chloride. The reaction mixture was cooled to -5°using a salt/ice bath. Then quickly added methanesulfonamide (51,15 g, 0,447 mol)followed by slow dropwise added triethylamine (54,37 g) in 225 ml of methylene chloride. Adding that requires ˜30 minutes, it was done so that the temperature of the reaction mixture never rose above about 5°C. After the addition the stirring was continued for 1 hour and the contents of the reactor was transferred into a 12-liter separating funnel, to which was added 2100 ml of methylene chloride. This solution is then washed with 700 ml aliquot of cold 1N HCl, 1N NaOH and saturated NaCl solution (each). Water washing were combined and subjected to back extraction with 3500 ml of methylene chloride. All organic washings were combined into 9 l of the vessel, which was added to the 500 g of neutral aluminum oxide with activity classes II and 500 g of anhydrous sodium sulfate. The contents of this container are thoroughly mixed for 30 minutes and filtered. The filtrate was evaporated to dryness in vacuum to obtain a resinous yellow foam. This foam was dissolved in 350 ml of methylene chloride and added dropwise with stirring was added to 1800 ml of a simple ester. The rate of addition was adjusted so that about half of ether was added over 30 minutes. After adding about 750 ml product began to stand out in the form of a crystalline solid. The remaining amount of 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°during the night, resulting in a received 144,61 g (88%) of nearly white solid, TPL 149-150°C. the Product thus obtained had usually 98-99% purity, as shown by HPLC (% area). In one cycle was obtained a product having a melting point 153-153, 5°With 99.5%purity, as determined by HPLC-square.

1H-NMR (CDCl3): USD 5.76(1H, s), is 5.18(1H, dt), 3,68(3H, s), 3,06(3H, s), 2,85(1H, m), 2,75-1,6(N, m), USD 1.43(3H, s)of 1.07(3H, s).

Example 24.

Scheme 1: stage 3C: Method a: Obtain 7-Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone

1-Liter chetyrehkolka the flask was equipped as described in the second example. In this flask with stirring and in the atmosphere of nitrogen was added formic acid (250 ml) and acetic anhydride (62 ml). Then add the potassium formate (6,17 g) and the reaction mixture was heated in an oil bath to an internal temperature of 40°With (this procedure was repeated at 70°With better results) for 16 hours. After 16 hours was added mesilate and the internal temperature was raised to 100°C. Heating and stirring were continued for 2 hours after which the solvent was removed in vacuum on a rotary evaporator. The residue was stirred with 500 ml of ice-cold water for fifteen minutes, and then twice was extracted with 500 ml aliquot of ethyl acetate. The organic phases were combined and then washed with 250 ml aliquot of cold saturated solution of sodium chloride (twice), 1N-sodium hydroxide solution and again with saturated sodium chloride. Then the organic phase was dried with sodium sulfate, filtered and concentrated to dryness in a vacuum, resulting in the obtained yellowish-white foam, which was applied by spraying on the glass with a putty knife. Analysis 14,65 g of the obtained powder (% area HPLC) indicated that the powder was a mixture 82,1% 7 Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone; 7,4% 7 Metelitza-17α-hydroxy-3-ekspresy-4,11 - Dien-7α,21-in primary forms, γ-lactone; and 5.7% 9α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid is, bis(γ-lactone).

1H-NMR (CDCl3): 5,74(1H, s), 5,67(1H, m), 3,61(3H, s)of 3.00(1H, m), 2,84(1H, DDD, J=2,6, 15), 2,65-2,42(6N, m), 2,3-2,12(5H, m), 2.05 is-1,72(4H, m), 1,55-to 1.45(2H, m)of 1.42(3H, s)to 0.97(3H, s).

Example 25.

Scheme 1: stage 3C: Method: Getting Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone

A 5-liter chetyrehosnuju flask, which was equipped as described above was added under stirring in nitrogen atmosphere 228,26 g of acetic acid and 41,37 g of sodium acetate. Using an oil bath and the mixture was heated to an internal temperature of 100°C. Then added mesilate (123,65 g) and heating was continued for another thirty minutes. At the end of this time, heating was discontinued and was added 200 ml of ice water. The temperature was lowered to 40°and continued to stir for another 1 hour, after which the reaction mixture was slowly poured into 1.5 liters of cold water in a 5-liter stir the flask. The product was isolated in the form of a resinous oil. This oil was dissolved in 1 l of ethyl acetate and washed with 1 l of cold saturated solution of sodium chloride, 1N 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 obtain foam, which was settled with the formation of resinous oil. This oil is triturated with simple ether is m for some period of time and it in the end, aterials. The obtained solid was filtered and washed with more ether to obtain 79,59 g yellow-white solid. This substance is comprised 70.4% of the target complex Δ9,11-tafira 6; 12,3% complex Δ11,12-tafira 8; 10,8% 7-α,9-α-lactone 9 and 5.7% of unreacted compounds 5.

Example 26.

Scheme 1: stage 3D: Method a: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

A 4-necked 500 ml reactor with jacket was equipped with a mechanical stirrer, fridge/bubbler, thermometer and addition funnel with a tube for supplying nitrogen. In this reactor with stirring in nitrogen atmosphere was loaded 8,32 g crude complex tafira in 83 ml of methylene chloride. After that was added was 4.02 g of dibasic potassium phosphate, and then 12 ml of trichloroacetonitrile. Through the jacket waterproof for external cooling and the reaction mixture was cooled to 8°C. In the addition funnel over 10 minutes was added 36 ml of 30% hydrogen peroxide. After complete addition, the reaction mixture initially having a pale yellow color, became almost colorless. During the addition the reaction mixture is maintained at a temperature of 9±1°and was stirred over night (a total of 23 hours). To the reaction mixture were added methylene chloride (150 ml) and the f content was added in ˜ 250 ml of ice water. This mixture was extracted with three times 150 ml aliquot of methylene chloride. United methylenechloride extracts were washed with 400 ml of cold 3% solution of sodium sulfite to decompose any residual peroxide. Then washed 330 ml cold wash with 1N sodium hydroxide, 400 ml of cold wash with 1N hydrochloric acid and, finally, washing with 400 ml of saturated salt solution. 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 vacuum to obtain 9,10 g crude product as a pale yellow solid. This substance was recrystallized from ˜25 ml of 2-butanone obtaining 5,52 g of almost white crystals. After the final recrystallization from acetone (˜50 ml) was received and 3.16 g of long needle-like crystals TPL 241 to 243°C.

1H-NMR (CDCl3): of 5.92(1H, s)to 3.67(3H, s), 3,13(1H, d, J=5), 2,89(1H, m), 2,81-2,69(15 NM, m)1,72(1H,DD, J=5,15), 1,52-1,22 (5H, m), 1.04 million(3H, s).

Example 27.

Scheme 1: step 3: Option 1: Conversion of 4'S(4'α),7'α-hexadecagon-11'α-hydroxy-10'β,13'β-dimethyl-3',5,20'-three-oxaspiro[furan-2(3H),17'β(4,7)-methane[17H]cyclopent[a]phenanthrene]-5'β(2 N)-carbonitrile in Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone.

In a clean and dry reactor was loaded on the ketone (20 g), and then added 820 ml Meon, and 17.6 ml of 25% solution of NaOMe/MeOH. The reaction mixture was heated under reflux (˜67°C) for 16-20 hours. Product extinguished 40 ml of 4n HCl. The solvent was removed at atmospheric pressure by distillation. Then was added 100 ml of toluene and the residual methanol was removed by azeotropic distillation with toluene. After concentration the crude complex hydroxyether 4 was dissolved in 206 ml of methylene chloride and cooled to 0°C. thereafter was added methanesulfonamide (5 ml)and then slowly added to 10.8 ml of triethylamine. This product was stirred for 45 minutes. The solvent was removed by vacuum distillation and received raw mesilate 5.

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

A mixture containing formic acid, was added to the concentrated solution 5 obtained as described above. This mixture was heated to 95-105°C for 2 hours. The mixture containing the product was cooled to 50°and volatile components were removed by vacuum distillation at 50°C. the Product was distributed between 275 ml of ethyl acetate and 275 ml of water. The aqueous layer was subjected to back extraction 137 ml of ethyl acetate, washed with 240 ml of cold 1N solution of sodium hydroxide, and then 120 ml of saturated NaCl. Th is phase separation the organic layer was concentrated by vacuum distillation to obtain crude complex enefer.

The product was dissolved in 180 ml of methylene chloride and cooled to 0-15°C. thereafter was added 8,68 g dailybeast, and then to 2.9 ml of trichloroacetonitrile. To the resulting mixture for 3 minutes was added 78 ml of 30% hydrogen peroxide. The reaction mixture was stirred for 6-24 h at 0-15°C. After passing the two-phase reaction mixture was left for phase separation. The organic layer was washed 126 ml of 3% solution of sodium sulfate, 126 ml 0,5N sodium hydroxide solution, 126 ml of 1N hydrochloric acid and 126 ml of 10% saturated salt solution. The product was dried with anhydrous sodium sulfate or filtered on celite, after which the solvent is methylene chloride was removed by distillation at atmospheric pressure. Product two times was led from ethyl ketone was obtained 7.2 g epoxyoctane.

Example 28.

Scheme 1: step 3: Option 2: Conversion of 1'S(4'α),7'α-hexadecagon-11'α-hydroxy-10'β,13'β-dimethyl-3',5,20'-three-oxaspiro[furan-2(3H), 17'β(4,7)-methane[17H]cyclopent[a]phenanthrene]-5'β(2 N)-carbonitrile in Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone, without the isolation of intermediate compounds.

A 4-necked 5 l round-bottom-flask was fitted with mechanical stirrer, addition funnel with a tube for nitrogen, a thermometer and a hole is dildocam with bubbler, attached to the scrubber with sodium hypochlorite. Into a flask of 3.05 l of methanol was added diketone (to 83.20 g). In the addition funnel was loaded 67,85 g 25% (wt./mass.) solution of sodium methoxide in methanol. Under stirring in nitrogen atmosphere in the flask for 15 minutes was added dropwise methoxide. Which resulted in formation of a dark orange/yellow suspension. The reaction mixture was heated under reflux for 20 hours and was added dropwise 175 ml of 4n hydrochloric acid while heating under reflux. (Caution during this reaction is allocated HCN!). The reflux condenser was replaced by the head nozzle with drain and 1.6 l of methanol was removed by distillation, dobavlyaya dropwise through the funnel 1.6 l of aqueous 10% solution of sodium chloride in accordance with the speed of distillation. The reaction mixture was cooled to room temperature and was extracted twice with 2.25 l aliquot of methylene chloride. The combined extracts were washed with cold 750 ml aliquot 1N sodium hydroxide and saturated sodium chloride solution. The organic layer was dried by azeotropic distillation with methanol under a pressure of one atmosphere to a final volume of 1 liter (0.5% of the total product was taken for analysis).

The concentrated organic solution (complex hydroxyether) was again added to the original reaction flask, najednou as described previously, but without traps for HCN. The flask was cooled to 0°and added a 30.7 g methanesulfonanilide, stirring in nitrogen atmosphere. In an addition funnel over 15 minutes dropwise introduced 32,65 g of triethylamine, maintaining the temperature at 5°C. the Stirring was continued for 2 hours, the reaction mixture was warmed to room temperature. Then prepared a column containing 250 g of acidic ion-exchange resin Dowex 50 W × 8-100, and before that, it was washed with 250 ml water, 250 ml of methanol and 500 ml of methylene chloride. The reaction mixture was removed from the column and collected. Then cooked fresh column and the above procedure was repeated. After that was preparing a third column containing 250 g of a basic ion-exchange resin Dowex 1 × 8-200 and pre-treated as a column with an acidic resin described above. The reaction mixture was removed from the column and collected. Then prepared the fourth column with ion exchange resin and the reaction mixture was again removed and collected. Then through each column missed two 250 ml portions methylenchloride leaching and each run required ˜10 minutes. The washing solvent was combined with the reaction mixture and concentrated in vacuo to a volume of ˜500 ml, and 2% of this volume was removed for quantification. Then the residue was concentrated to a final volume of 150 ml (crude rest the R nelfinavir).

In the initial 5-liter reaction apparatus was added to 960 ml of formic acid, 472 ml of acetic anhydride and 23,70 g of potassium formate. This mixture was heated with stirring in nitrogen atmosphere to 70°C for 16 hours. Then the temperature was raised to 100°and within 30 minutes via an addition funnel was added to the crude solution of nelfinavir. As the distillation of the methylene chloride from the reaction mixture, the temperature was lowered to 85°C. After all the acid was removed, the temperature was again increased to 100°and it was maintained for 2.5 hours. The reaction mixture was cooled to 40°and formic acid was removed under pressure to achieve a minimum amount of mixing (˜150 ml). The residue was cooled to room temperature and was added to 375 ml of methylene chloride. Dilute the residue was washed with cold 1-liter portions of saturated sodium chloride solution, 1N sodium carbonate and the sodium chloride solution. The organic phase was dried by magnesium sulfate (150 g) and filtered to obtain a dark reddish-brown solution (solution of the crude complex enefer).

4-Necked 1-liter jacketed reactors supplied with a mechanical stirrer, fridge/bubbler, thermometer and addition funnel with a tube for supplying nitrogen. The reactor was loaded crude solution of complex enefer (approx the tion 60 g) in 600 ml of methylene chloride with stirring in nitrogen atmosphere. After this was added 24,0 g dibasic potassium phosphate, and then 87 ml trichloroacetonitrile. Through the jacket of the reactor was supplied water for external cooling and the reaction mixture was cooled to 10°C. This mixture for 30 minutes was added to the addition funnel with 147 ml of 30% hydrogen peroxide. When you are finished adding originally painted a dark reddish-brown color of the reaction mixture had become light yellow in color. Throughout the period of addition, the reaction mixture was maintained at 10±1°and the stirring continued overnight (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 210 ml of 3% solution of sodium sulfite. This procedure was repeated once more, after which the organic and aqueous part was found negative reaction to peroxide, as evidenced by the indicator paper with starch/iodide. The organic phase is then washed 210 ml aliquot of cold 1N sodium hydroxide, 1N hydrochloric acid and, finally, two washings with saturated salt solution. The organic phase was dried by azeotropic distillation to a volume of ˜100 ml, then added fresh solvent (250 ml) and subjected to azeotropic distillation is similar to ˜100 ml and the remaining solvent was removed the vacuum obtaining 57,05 g crude product as yellow resinous foam. Then part (51,01 g) the product was dried to constant weight 44,3 g and quantitatively assessed using HPLC. The analysis indicated 27.1% epoxyoctane.

Example 29. Obtaining 3-ethoxy-11α-hydroxy-androsta-3,5-Dien-17-she's from 11α-hydroxyandrostenedione

Into the reaction flask under nitrogen atmosphere was introduced 11α-hydroxyandrostenedione (of 429.5 g) and hydrate toluensulfonate acid (7,1). To the reactor was added ethanol (2,58 l) and the resulting solution was cooled to 5°C. In this solution for 15 minutes was added triethylorthoformate (334,5 g) at a temperature of from 0°to 15°C. After complete addition, triethylphosphate the reaction mixture was heated to 40°and was left for reaction at this temperature for 2 hours, after which the temperature was raised up to the temperature of heating under reflux, and the reaction continued at reflux for a further 3 hours. The reaction mixture was cooled under vacuum and the solvent was removed in vacuo to obtain 3-ethoxy-11α-hydroxyandrost-3,5-Dien-17-it.

Example 30. Getting enamine of 11α-hydroxykynurenine

Scheme 1: step 1: Method: Obtain 5'R(5'α),7'β-20'-aminohexanoate-11'β-hydroxy-10'α,13'α-dimethyl-3',5-dioxaspiro[furan-2(3H),17'α(5 N)-[7,4]metheno[4H[cyclopent[a]phenanthrene]-5'-carbonitrile.

Cyanide NAT the Oia (1,72 g) were placed in a 25 ml 3-necked flask, equipped with a mechanical stirrer. Then added water (2.1 ml) and the mixture was stirred while heating up until the solids dissolved. After this was added dimethylformamide (15 ml), and then 11α-hydroxybenzene (5.0 g). To the mixture was added a mixture of water (0.4 ml) and sulfuric acid (1,49 g). This mixture was heated to 85°C for 2.5 hours, for which HPLC analysis showed complete conversion to product. The reaction mixture was cooled to room temperature. Then was added sulfuric acid (0,83 g) 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 3 ml of DMF and 5 ml of water. The suspension was stirred for 40 minutes and filtered. The residue on the filter is washed twice with 40 ml of water and dried in a vacuum oven at a temperature of 60°during the night with 11α-hydroxylamine, ie 5'R(5'α),-7'β-20'-aminohexanoate-11'β-hydroxy-10'α,13'α-dimethyl-3',5-dioxaspiro[furan-2(3H),-17'α(5 N)-[7,4]metheno[4H]cyclopent[a]phenanthrene]-5'-carbonitrile (4.9 g).

Example 31. The transformation of 11α-hydroxykynurenine in diketone in one tank

In a 50 ml 3-necked flask equipped with a mechanical stirrer, was added sodium cyanide (1,03 g). After adding water (1,26 ml) the flask was gently heated to dissolve the firm is about substance. Then was added dimethylacetamide [or dimethylformamide] (9 ml), then 11α-hydroxybenzene (3.0 g). Into the reaction flask with stirring was added a mixture of sulfuric acid (0,47 ml) and water (0.25 ml). The resulting mixture was heated to 95°C for 2 hours. HPLC analysis indicated completion of reaction. Then was added sulfuric acid (0,27 ml) and the mixture was stirred for 30 minutes. After this has introduced additional amount of water (25 ml) and sulfuric acid (0,90 ml) 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 filter of sintered glass, followed by a double rinse with water (20 ml). Solid diketone, ie 4'S(4'α),7'α-hexadecagon-11'α-hydroxy-10'β,13'β-dimethyl-3',5-20'-dioxaspiro[furan-2(3H),-17'β(4,7)methane[17H]cyclopent[a]phenanthrene]-5'β(2 N)-carbonitril, dried in a vacuum oven to obtain 3.0 g of a solid substance.

Example 32A-1.

Scheme 1: stage 3A: Method: Getting Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

A suspension of 5.0 g of the diketone obtained by the method described in Example 31, in methanol (100 ml) was heated under reflux and within 1 minute was added a 25% solution of potassium methoxide in methanol (5.8 ml). The mixture became homogeneous. After 15 mi the ut precipitate appeared. The mixture was heated under reflux and after about 4 hours it was again homogeneous. The reflux was continued full 23.5 hours and was added 4.0 n HCl (10 ml). All 60 ml of a solution of hydrogen cyanide in methanol was removed by distillation. To the residue obtained by distillation, for 15 minutes was added water (57 ml). During the addition of water the temperature of the solution was raised to 81,5°and With another 4 ml of a solution of hydrogen cyanide/methanol was removed by distillation. After the addition of water was complete, the mixture became turbid and the heating source was removed. The resulting mixture was stirred for 3.5 hours and the product slowly crystallized. The suspension was filtered and the collected solid is washed with water, dried in a stream of air in the funnel, and then dried at 92° (26 inches Hg) for 16 hours to obtain 2,98 g of a whitish solid. This solid substance was a 91,4% complex hydroxyether, i.e. Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-carboxylate, γ-lactone (by weight). The output amounted to 56.1%.

Example 32A-2. Getting Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

Diketone (40 g)obtained by the method described in Example 31, was loaded in a clean, dry 1-liter reactor with what ubashi, equipped with a bottom drain outlet, refrigerator, RTD sensor and a collector for collecting fractions. In this reactor was loaded methanol (800 ml) and the mixture was stirred. The resulting suspension was heated to 60-65°and added a 25% solution of potassium methoxide (27.8 ml). The mixture became homogeneous.

The resulting mixture was heated under reflux. After about 1.5 hours of heating under reflux the mixture at reflux was added and 16.7 ml of 25% solution of potassium methoxide. The mixture was maintained at reflux for another 6 hours. The conversion of the diketone in complex hydroxyether analyzed using HPLC. After HPLC analysis showed the ratio of diketone complex hydroxyether less than about 10%, to the mixture for about 15 minutes while heating under reflux was added 77 ml of 4M HCl (hydrochloric acid may be replaced by a comparable number 1,5M-3M sulfuric acid).

Then the mixture was subjected to distillation and about 520 ml of distillate methanol/HCN was collected and discarded. The concentrated mixture was cooled to about 65°C. To the mixture for 90 minutes was added about 520 ml of water and during the addition the temperature was maintained at about 65°C. the Mixture was gradually cooled to about 15°With approximately four hours, and then stirred and support is supported at a temperature of about 15° Even for two hours. The mixture was filtered and the filtered product twice washed with 200 ml of water each time). The filtered product was dried in vacuum (90°C, 25 mmHg). Received approximately 25-27 g of a whitish solid substances containing mainly Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone.

Example 32V-1. Getting 7 Metelitza-5β-cyano-11α,17-dihydroxy-3-oxo-17α-pregnan-7α,21-in primary forms, γ-lactone

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

1H-NMR (CDCl3) of 0.95 ppm(s, 3H), 1,4(s, 3H), 3,03(d, 1H, J=15), of 3.69(s, 3H), 4,1(m, 1H).

13C-NMR (CDCl3) 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,, 45,7, 47,5, 52,0, 68,0, 95,0, 121,6, 174,5, 176,4, 207,0.

Example 32V-2. Getting 7 Metelitza-5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α,21-in primary forms, γ-lactone

To 2.0 g (4,88 mol) 9,11-epoxidation formula 21, suspended in 30 ml of anhydrous methanol, was added to 0.34 ml (2.4 mmol) of triethylamine. The suspension was heated under reflux and after 4.5 hours was not observed in the presence of the source material, as it was shown by HPLC (Bond SB-C8, 150 × 4.6 mm, 2 ml/min, linear gradient of 35:65:45:55 And:within 15 minutes, A=acetonitrile/methanol 1:1, A=water/0.1% of triperoxonane 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 and was obtained 1.3 g of 7-Metelitza-5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α,21-in primary forms, γ-lactone, in the form of a white solid. The filtrate was concentrated to dryness on a rotary evaporator, and the residue was washed with 3-5 ml of methanol. After filtering received another 260 mg 7 Metelitza-5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α,21-in primary forms, γ-lactone. The output amounted to 74.3 per cent.

1H NMR (400 MHz, deuterochloroform) δ: 1,00(s, 3H), of 1.45 (m, 1H), 1,50(s, 3H), of 1.65(m, 2H), 2,10(m, 2H), 2,15-to 2.65 (m, 8H), 2,80(m, 1H), 2,96(m, 1H), 3,12(d, J=13, 1H), 3,35(d, J=7, 1H), to 3.67 (s, 3) - Rev.).

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

In the reaction flask was loaded 6.8 g of the diketone (obtained by the method described in Example 31), 68 ml of acetonitrile, 6.0 g of sodium acetate and 60 ml of water. The mixture was heated and stirred under reflux. After 1.5 hours the mixture became almost homogeneous. After 3 hours was added 100 ml of water, at least distillation 50 ml of acetonitrile. 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 reduce the pH to about 4.5 and a solid substance was precipitated. The solid was isolated, washed with water and dried, resulting in a received 4.5 g 5β-cyano-11-α,17-dihydroxy-3-oxo-17α-pregnan-7α,21-dicarboxylic acid, γ-lactone.

1H-NMR (DMSO) ppm 0,8(C, H), 1.28(in, N), 3,82(m, 1H).

13C-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: Getting Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Diketone (30 g)obtained by the method described in Example 31, was loaded into the cleaned and dried 3-necked reaction flask equipped with a thermometer, a trap Dina-Starkey mechanical stirrer. Methanol (24 ml) were loaded into the reactor at room temperature (22° (C) and the resulting suspension was stirred for 5 minutes. The reactor was loaded 25% of the mass. solution of sodium methoxide in methanol (52,8 ml) and the resulting mixture was stirred at room temperature for 10 minutes, during which time the reaction mixture turned into a light brown clear solution was observed, a small temperature increase (2-3°). The rate of addition was regulated to prevent temperature increase in the tank over 30°C. Then the mixture was heated under reflux (about 67° (C) and continued heating under reflux for 16 hours. Then took the sample and analyzed by HPLC for the transformation. The reaction mixture continued to heat under reflux until then, until the level of residual diketone did not exceed 3 percent of the diketone. While heating under reflux in the reaction vessel was loaded 4n HCl (120 ml), which was formed HCN, which was suppressed in the scrubber.

After the reaction from the reaction mixture drove 90-95% methanol solvent at atmospheric pressure. The temperature at the head of the distillation process ranged from 67 to 75°and distillate, which contained HCN, was treated with caustic soda and bleach, and the eat utilitybase. After removal of methanol, the reaction mixture was cooled to room temperature and while cooling the mixture in the range of 40-45°began to precipitate the solid product. Aqueous solution, optionally containing 5% wt. sodium bicarbonate (1200 ml) at 25°C, loaded into a chilled suspension, and then the mixture was cooled to 0°With approximately 1 h 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, Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone, was isolated by filtration and the filter cake washed with water (100 ml). The product was dried at 80-90°in vacuum under a pressure of 26" Hg (inches MRT.) to constant weight. The water content after drying was less than 0.25% of the mass. Adjusted molar yield was within 77-80% of the mass.

Example 34.

Scheme 1: stage 3A: Method D: Getting Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Diketone obtained by the method described in Example 31 (1 EQ.), was subjected to reaction with sodium methoxide (4.8 EQ.) in a methanol solvent in the presence of zinc iodide (1 EQ.). The processing of the product is conducted either way, providing for the extraction and described in this application in any way without extraction, which did not contain any stage extraction with methylene chloride, washing with a saturated saline solution and caustic soda and drying with sodium sulfate. In addition, in the method, not involving the extraction, the toluene was replaced by 5% of the mass. the sodium bicarbonate solution. The quality of the product was identified Metelitza-11α,17α-dihydroxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone.

Example 35.

Scheme 1: stage 3C: Method: Getting Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone

Complex hydroxyether, obtained as described in Example 34 (1.97 g)was combined with tetrahydrofuran (20 ml) and the resulting mixture was cooled (-70°). Then add sulfurylchloride (0.8 ml) and the mixture was stirred for 30 minutes, after which was added imidazole (1.3 g). The reaction mixture was heated to room temperature and stirred for another 2 hours. Then the mixture was diluted with methylene chloride and was extracted with water. The organic layer was concentrated to obtain the crude product, Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone (1.97 g). A small sample of the crude product was analyzed by HPLC. The analysis showed that the ratio of the 9,11-olefin:11,12-olefin:7,9-the Acton was 75.5:7,2:17,3. If the reaction was carried out at 0°or otherwise, as described above, this reaction gave a product in which the ratio of the 9,11-olefin:11,12-olefin:7,9-lactone was 77,6:6,7:15,7. This procedure was combined in one stage, the introduction of the leaving group, and removing it for the subsequent introduction 9,11-olefin structure of the complex of tafira, i.e. the reaction with sulfurylchloride leads to the replacement of 11α-hydroxy-group of complex hydroxyether formula V) halide and subsequent dehydrohalogenation education Δ9,11-patterns. Thus, the formation of complex tafira proceeds effectively without the use of strong acids (such as formic acid or desiccant, such as acetic anhydride. Also excluded stage of heating under reflux, carried out in an alternative method, which produces carbon monoxide.

Example 36A.

Scheme 1: stage 3C: Method D: Getting Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone.

Complex hydroxyether (20 g), obtained as described in Example 34, and methylene chloride (400 ml) was added to the purified dry three-neck round-bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple. The resulting mixture was stirred at room temperature till complete education solution. The solution which was gladly to 5° With the use of ice baths. To a solution of CH2Cl2containing complex hydroxyether, added methanesulfonamide (5 ml)and then dropwise slowly added triethylamine (10,8 ml). 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 analysis), the mixture was concentrated at about 0°under pressure of 26 inches of Hg until then, until it formed a thick slurry. The resulting suspension was diluted with CH2Cl2(160 ml) and the mixture was concentrated at about 0°under pressure of 26 inches of Hg to obtain a concentrate. It was found that the purity of the concentrate (mutility product of the formula IV, where R3=H and-And-And - and-In-To - represent-CH2-CH2-i.e. the transformation of Metelitza-11α,17α-dihydroxy-3-oxo-pregn-4-ene-7α,21-in primary forms, γ-lactone in Metelitza-17α-hydroxy-11α-(methylsulphonyl)oxy-3-oxoprop-4-ene-7α,21-di-carboxylate, γ-lactone) is 82% (% area HPLC). This material was used in the next reaction without isolation.

In a clean dry reactor equipped with a mechanical stirrer, a refrigerator, a thermocouple and a casing for heating, was added to form the t potassium (4.7 g), formic acid (16 ml) and acetic anhydride (8 ml, 0,084 mol). The resulting solution was heated to 70°and was stirred for about 4-8 hours. The addition of acetic anhydride resulted in an exothermic reaction and the formation of gas (CO), and therefore to control the temperature and gas (pressure) the speed of adding appropriately corrected. The reaction time for production of the active elimination reagent is dependent on the amount of water present in the reaction (formic acid and potassium formate contained about 3-5% of the water each). The elimination reaction is sensitive to the amount of water present; that is, if this number >0,1% water (KF), the level of the 7,9-lactone impurity can be increased. This byproduct is difficult to remove from the final product. If KF is <0.1% of water in the concentrate nelfinavir (0,070 mol)obtained in the preceding stage, introduced eliminating active agent. The resulting solution was heated to 95°and volatile material were released and collected in the trap Dean-stark. At the termination of the emission of volatile material trap Dean-stark replaced the fridge and the reaction mixture was heated for 1 hour at 95°C. after this time TLC and HPLC analysis; and <0.1% of the source material), the contents were cooled to 50°and began disti is the transmission in the vacuum of 26 inches of Hg/50° C). The mixture was concentrated to a thick slurry and then cooled to room temperature. The resulting suspension was diluted with ethyl acetate (137 ml) and the solution was stirred for 15 min and then was diluted with water (137 ml). The layers were separated and the aqueous layer was again extracted with ethyl acetate (70 ml). United an ethyl acetate solution once washed with saturated saline solution (120 ml) and twice chilled on ice, 1N NaOH solution (120 ml each). the pH of the aqueous layer was measured, and if the pH of the spent wash was <8, the organic layer was again washed. If the pH of the spent wash was >8, an ethyl acetate layer was once washed with salt solution (120 ml) and concentrated to dryness on a rotary evaporator using a water bath 50°C. as a result received 92 g of complex tafira, solid product, i.e. Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone (exit 77% mol).

Example 36V. Getting Metelitza-17α-hydroxy-3-oxo-pregna-4,9(11)-Dien-7α,21-in primary forms, γ-lactone.

In a clean, dry three-neck 250 ml round bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple was added 25 g (53,12 mmol) of complex hydroxyether Metelitza-11α,17α-dihydroxy-3-ekspresy-4-ene-7α,21-in primary forms, γ-lactone, extracting the aqueous as described in Example 34, and then 150 ml of methylene chloride (Burdick & Johnson). The resulting mixture was stirred at room temperature until, until there was obtained a light suspension. The solution was cooled to -5°in the bath with ice. To a solution of methylene chloride containing complex hydroxyether, added methanesulfonamide (7,92 g, 69,06 mmol) (Aldrich), and then immediately dropwise slowly added triethylamine (7,53 g) (Aldrich). The rate of addition was adjusted so that the reaction temperature did not exceed 0°C. This reaction was very exothermic; therefore, she needed to cool down. Add accounted for 35 minutes. The reaction mixture was stirred at about 0°With a further 45 minutes. At the completion of the reaction (when she was less than 1% of the hydroxyether, as it was shown by HPLC and TLC analysis), the mixture was concentrated by evaporation to approximately 110-125 ml methylenchloride solvent at atmospheric pressure. During evaporation, the reaction temperature reached approximately 40-45°C. If after 45 minutes the reaction was completed, the reactor can be loaded even 0.1 equivalent of methanesulfonanilide and another 0.1 equivalent of triethylamine with subsequent estimation of the reaction to completion. The resulting mixture contained the crude product Metelitza-17α-hydroxy-11α-(methylsulphonyl)oxy-3-oxoprop-4-ene-7α,21, in primary forms, γ-lactone. This is the product was used in subsequent reactions without highlighting.

A second 250-ml clean and dry reactor equipped with a mechanical stirrer, a refrigerator, a thermocouple and a casing for heating, was added anhydrous sodium acetate (8,7 r)(Mallinkrodt), glacial acetic acid (42,5 ml)(Fisher) and acetic anhydride (0.5 ml)(Fisher). The resulting solution was heated to 90°and was stirred for about 30 minutes. Since the addition of acetic anhydride is accompanied by a temperature increase, the addition was carried out at such speed that it corrected as 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 was pointing to <0.1% of water, the acetic acid solution was transferred into a concentrate nelfinavir obtained, as reported in the first section of this example. After migration, the temperature of the mixture ranged from about 55°C to 60°C. the total 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 this temperature for about 60-90 minutes until until stopped volatile substances. Volatile substances, warded off from the mixture, was collected in the trap Dean-stark. After completion of the reaction (TLC and HPLC analysis; and <0.1% of the original substance) source n is grovania removed. When the temperature of the mixture reached 80°to this mixture within 60-90 minutes was slowly added 150 ml of water. After adding water, the mixture was cooled to a temperature of from about 35°C to 45°and began to form a suspension. 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 water. Then the filtrate was washed a second time with a further 100 ml of water. The obtained filtrate was dried at 70°in vacuum with the receipt of 25.0 g of dry anafinova product, Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, 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 good results (compared with similar ways, namely: (i) reduces the volume of solvents, (ii) reduces the number of individual working stages required to produce complex of tafira of complex hydroxyether, (iii) avoids the need for leaching, (iv) the allocation of the final product allows you to replace the extraction precipitation with water; and (v) provides a reliable elimination, which previously was associated with the formation of the mixed anhydride and the release of gas when using formic acid instead of the UKS the Noah acid.

Example 37A.

Scheme 1: stage 3C: Method E: Getting Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone.

In a three-neck 2 l round bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple was added difficult hydroxyether (100 g, 0.22 mol). Bath with circulating cooling was equipped with an automatic temperature controller. Before beginning the reaction flask was drained due to the sensitivity of methanesulfonanilide to the water.

Into the flask was loaded methylene chloride (1 l) and dissolved therein a complex hydroxyether by stirring. The solution was cooled to 0°and into the flask through the addition funnel was loaded methanesulfonanilide (25 ml; 0.32 mol). Into the reaction flask through the addition funnel was loaded triethylamine (50 ml, 0.59 mol) and the funnel was rinsed with additional quantity of methylene chloride (34 ml). Adding triethylamine was highly exothermic. Time added was about 10 minutes under stirring and cooling. Downloaded the mixture was cooled to 0°C and held at this temperature while stirring for another 45 minutes, during which the head region of the reaction flask was purged with nitrogen. The sample was then the reaction mixture was analyzed using thin-layer chromatography and high-performance liquid chromatography for monitoring the progress of R the shares. After that, the mixture was stirred at 0°With a further 30 min and again evaluated on the completion of the reaction. The analysis showed that at this stage the reaction was mostly complete; methylenchlorid the solvent is evaporated at 0°in vacuum at 26" Hg analysis of the distillate by gas chromatography indicated the presence of methanesulfonamide and triethylamine. Then the reaction flask was loaded methylene chloride (800 ml) and the resulting mixture was stirred for 5 minutes at a temperature in the range of 0-15°C. then the solvent is again evaporated at 0-5°under pressure 26" Hg and received mesilate formula IV, where R3is N; -a-a - and-B-To-represent-CH2-CH2-, a R1is methoxycarbonyl. The purity of the product was about 90-95% (by area).

To obtain eliminating reagent potassium formate (23,5 g; 0.28 mol), formic acid (80 ml) and acetic anhydride (40 ml) was mixed in a separate drained reactor. Formic acid and acetic anhydride was applied to the reactor pump and during the addition of acetic anhydride, the temperature was maintained at a level of not more than 40°C. a Mixture of eliminating reagent was heated to 70°to divert water from the reaction system. This reaction was continued up until the water content was lower than 0.3 wt. -%, as was measured by Ana the AIA Karl Fischer. Then a solution of the reagent for the elimination transferred to the reactor containing the concentrated crude solution nelfinavir, obtained as described above. The resulting mixture was heated to the maximum temperature 95°and volatile distillate was collected until, when the distillate was not formed. Distillation was stopped when about 90°C. After distillation of the reaction mixture was stirred at 95°another 2 hours and the completion of the reaction was monitored using thin-layer chromatography. When the reaction was completed, the reactor was cooled to 50°and formic acid and the solvent was removed from the reaction mixture in vacuum under a pressure of 26" Hg at 50°C. the Concentrate was cooled to room temperature, and then injected acetate (688 ml) and a mixture of ethyl acetate and concentrate was stirred for 15 minutes At this stage entered 12% salt solution (688 ml) to facilitate the removal of the organic phase of the water-soluble impurities. The phases were left for 20 minutes to precipitate. The aqueous layer was transferred into another vessel, which was loaded with more ethyl acetate (350 ml). This back extraction of the aqueous layer was carried out for 30 minutes, after which the phases were left to precipitate, and an ethyl acetate layers were combined. It combined an ethyl acetate layer was added a saturated solution of sodium chloride (600 ml) and p is remesiana was carried out for 30 minutes. Then the phases were left to precipitate. The aqueous layer was removed. Then there was an additional washing with sodium chloride (600 ml). The organic phase is separated from the second exhaust wash solution. After that, the organic phase is washed with 1N sodium hydroxide (600 ml) with stirring for 30 minutes. Phase besieged for 30 minutes to remove the water layer. After evaluation of the pH of the aqueous layer was discovered that he was >7. Additional washing with saturated sodium chloride (600 ml) was carried out for 15 minutes. And, finally, the organic phase was concentrated in vacuum at 26 mm Hg at 50°and the product was isolated by filtration. After drying, the final product was a brown foamy solid. This substance, in addition, were dried at 45°C under reduced pressure for 24 hours from the receipt of 95.4 g anafinova product, Metelitza-17α-hydroxy-3-ekspresy-4,9(11)-Dien-7α,21-in primary forms, γ-lactone, which was analyzed amounted to 68.8%. The molar yield was 74.4%, which was adjusted as the original complex hydroxyether and destination complex enefer.

Example 37V. Getting 7 Metelitza-17-methyl-3-oxo-18-norpregna-4,9(11),-13-triene-7α,21-in primary forms

In the reaction flask was loaded 5.5 g nelfinavir received the wow way, described in Example 23, 55 ml 94,3% formic acid and 1.38 g of potassium formate. The mixture was heated and stirred at reflux (104° (C) within two hours. After these two hours of formic acid, drove away under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10%potassium carbonate (50 ml). Selected water part was yellow in color. The ethyl acetate was washed with 5%sodium hydroxide (50 ml). Water parts were combined and acidified with diluted hydrochloric acid and nerastvorimaya substance was extracted with ethyl acetate. The ethyl acetate evaporated to dryness under reduced pressure to obtain 1.0 g of the residue, 7-Metelitza-17-methyl-3-oxo-18-norpregna-4,9(11),13-triene-7α,21-in primary forms.

1H-NMR (CDCl3) m:d: 1,5(s, 3H), 1,4(s, 3H), 3,53(s, 3H), 5,72(m, 1H).

13C-NMR (CDCl3) ppm: a 25.1 and 25.4(18 CH3and 19 CH3), 40,9 (10), 48,5(17)51,4(och3), 118,4(CH 11), and 125.4(4 CH), 132,4 (9), 138,5 and 139,7(13 C and 14 C), 168,2(5), 172,4(7), 179,6(22), 198,9(3).

Example S. Getting 7 Metelitza-5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-in primary forms, γ-lactone.

In the reaction flask was loaded 5.5 g nelfinavir, obtained as described in Example 23, 55 ml 94,3% formic acid and 1.38 g of potassium formate. The mixture was heated and stirred with a reflux is m (104° (C) within two hours. After these two hours of formic acid, drove away under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10% potassium carbonate (50 ml). Selected water part was yellow in color. The ethyl acetate was washed with 5% sodium hydroxide (50 ml). The ethyl acetate evaporated to dryness under reduced pressure to obtain 3.7 g of residue. Part (3.4 g) of the residue was chromatographically at 267 g of Merck silica gel (40-63 μm). The product was isolated with the scheme of elution by ethyl acetate and toluene (37:63)(on/about). After drying, this product was obtained 0,0698 g of the residue, 7-Metelitza-5β-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-in primary forms, γ-lactone.

1H-NMR (CDCl3) ppm: of 1.03(s, 3H), 1,22(s, 3H), 3,70(s, 3H), ceiling of 5.60(d, 1H, J=10), 5,98(d, 1H, J=10).

MICK cm-1: 2229 (CN), 1768 (lactone), 1710 (ester).

Example 37D. Selection 9α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid, bis(γ-lactone)

9α,17-Dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid, bis(γ-lactone) is a by-product elimination 11-nelfinavir. 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 residue were chromatographically 2.41 kg force is of Kagel Merck (40-63 microns) scheme for gradient elution with ethyl acetate and toluene (20:80, 30:70, 40:60, 60:40 about/about). Enriched mixture of 10.5 g) of the enamine and 7,9-lactone was obtained in fractions in the ratio of 60:40. The progress of purification was monitored by TLC on plates with EMF, elwira (60:40 V/V) ethyl acetate and toluene, and perform rendering using sulfuric acid, SWUV. Then part (10.4 g) of the mixture was purified using reverse-phase HPLC on a Kromasil C8 (7 μm) mobile phase: milliQ water and acetonitrile, 30:70 V/V. Of the mobile phase was separated 7,9-lactone of 2.27 g) as crystals.

MICK cm-1: 1762(7,9-lactone and 17-lactone), 1677, 1622(3-keto-Δ4,5).

1H-NMR (CDCl3) ppm: 1.00 and(s, 3H), 1,4(s, 3H), 2.05 is(d, 1H), 2,78(d, 1H), by 5.87 (s, 1H).

13C-NMR (CDCl3) 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: 71,85 and N 7,34; found: 71,68 and N 7,30.

Example E. Selection 7 Metelitza-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-in primary forms, γ-lactone

Connection 7 Metelitza-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21, in primary forms, γ-lactone was isolated after conducting multiple preparative liquid chromatography of the reaction mixture obtained after the reaction of elimination 11 mesilate-group (Example 24). It is a part of the accumulations of less polar impurities, as was assessed by TLC on plates with EMF using the m system elution with ethyl acetate and methylene chloride (30:70 V/V) and visualized by using sulfuric acid, SWUV. Basically, these less polar impurities were isolated from the crude enamine by preparative liquid chromatography. In particular, 9.6 g of crude raminosoa solution was chromatographically at 534 g of Merck silica gel (40-63 μm) using the scheme of gradient elution by ethyl acetate and toluene(20:80, 30:70, 40:60, 60:40 about/about). Less polar impurities were concentrated in the fractions in the ratio of 30:70. This way collected 12.5 g pool of less polar impurities. Then, this product has chromatographically 550 g of Merck silica gel (40-63 μm) using the scheme of gradient elution with ethyl acetate and methylene chloride(5:95, 10:90, 20:80, 30:70 about/about). Enriched part of the 20:80 fractions gave 1.2 g of residue. Additional chromatography 1.2 g of residue 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 about./about.) gave 0.27 g 7 Metelitza-5-cyano-17-hydroxy-3-oxo-17α-pregn-11-ene-7α,21-in primary forms, γ-lactone of the enriched part of the fractions of 10:90.

MS M+425 calculated for C25H31NO5(425,52).

MICK 2222 cm-1(nitrile), 1767 cm-1(lactone), 1727 cm-1(ester and 3-ketone).

1H-NMR (CDCl3) to 0.92 ppm(s, 3H), of 1.47(s, 3H), 2.95 and(m, 1H), the 3.65 (s, 3H), 5,90(m, 1H).

13C-NMR (CDCl3) of 14.0 ppm(18 CH3), 23.5cm(15 CH2), 27,0 (19 CH3) 37,8, 38,5 and 40.9(7,8 and 14 CH), 52,0(och3), 95,0(17), 121,5(23 CN) TO 123.5(CH 11), 135,3(9), 174,2 and 176,2 (22 and 24), 206(3).

Example 37F. Getting 7 Metelitza-17-hydroxy-3-oxo-11α-(2,2,2-Cryptor-1 oksidoksi)-17α-pregn-4-ene-7α,21-in primary forms

Complex hydroxyether (2.0 g, 4.8 mmol)obtained by the method described in Example 34, was added in 40 ml of methylene chloride, in a clean, dry 3-necked round bottom flask, equipped with a mechanical stirrer. Then the solution was added triethylamine (0,61 g, 6,10 mmol) and triperoxonane anhydride (1.47 g, 7.0 mmol). This mixture was stirred over night at room temperature.

The mixture is then diluted with 40 ml of methylene chloride. After the mixture is then washed with 40 ml of water, 40 ml of 1N HCl and 40 ml of 1N NaOH solution. Then the resulting solution was dried with magnesium sulfate and concentrated to dryness, which was obtained 3.2 g of a light brown solid, 7-Metelitza-17-hydroxy-3-oxo-11α-(2,2,2-Cryptor-1 oksidoksi)-17α-pregn-4-ene-7α,21-in primary forms.

The residue was analyzed and purified using chromatography. Conditions for HPLC: column Waters Symmetry C18 (150 mm × 4.6 mm votem.; the particle size 5 microns); temperature column - room; mobile phase : acetonitrile/water, 30/70 by volume; the pause flow 1.0 ml/min; volume of injection : 20 microlitres; the concentration of the sample - 1.0 mg/ml; detection UV at 210 nm; pressure - 1500 lb/to duim (105,5 kg/cm 2); and the running time is 45 minutes. Conditions for TLC: the adsorbent is silica gel Merck 60 F254; the system solvent - ethyl acetate/toluene, 65/35 by volume; visualisation technique - shortwave; and the number of application - 100 micrograms.

Example 37G. Getting 7 Metelitza-11α-(atomic charges)-17-hydroxy-3-oxo-17α-pregn-4-ene-7,21-in primary forms, γ-lactone

Complex hydroxyether (2.86 g, 6,87 mmol)obtained by the method described in Example 34, was added in 40 ml of methylene chloride, in a clean, dry 3-necked round bottom flask, equipped with a mechanical stirrer. Then the solution was added triethylamine (1.39 g, 13.7 mmol), dimethylaminopyridine (0.08 g, 0.6 mmol) and acetic anhydride (1,05 g of 10.3 mmol). This mixture was stirred over night at room temperature.

The mixture is then diluted with 150 ml ethyl acetate and 25 ml of water. Then an ethyl acetate solution is washed with 25 ml of citric acid solution. Then the resulting solution was dried with magnesium sulfate and concentrated to dryness, resulting in a received 3.33 g of light brown solid, 7-Metelitza-11α-(atomic charges)-17-hydroxy-3-oxo-17α-pregn-4-ene-7,21-in primary forms, γ-lactone.

Then the residue was analyzed and purified using chromatography. Conditions for HPLC: column Waters Symmetry C18 (150 mm × 4.6 mm votem.; the particle size of microns); the temperature of the column - room; mobile phase - acetonitrile/water 30/70 by volume; flow rate - 1.0 ml/min; volume of injection : 20 microlitres; the concentration of the sample - 1.0 mg/ml; detection UV at 210 nm; pressure - 1500 psi (105,5 kg/cm2); and the running time is 45 minutes. Conditions for TLC: adsorbent silica gel Merck 60 F254system ; the solvent is methylene chloride/methanol 95/5 by volume; visualisation technique - shortwave; and the number of application - 100 micrograms.

Example N.

Scheme 1: stage 3C: Method F: Obtain 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone

The potassium formate (1.5 g, 0.018 mol), formic acid (60 ml, 1.6 mol) and acetic anhydride (29.5 ml, 0.31 mol) was added in a clean, dry 250 ml reactor, equipped with a mechanical stirrer, a refrigerator, a thermocouple and a casing for heating. Then the solution was stirred 4 hours at 70°and cooled to room temperature, resulting in the received reagent for the elimination used to turn nelfinavir formula IV in the product of this example.

Prior eliminating the anhydride reagent TFA/TFA was added to 70,0 (0,142 mol) nelfinavir obtained by the method described in Example 23. The resulting mixture was heated to 95-105°C for 2.5 hours, the degree of conversion period is automatic controlled by 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 defend overnight at room temperature. The layers were separated and the aqueous phase was again extragonadal with ethyl acetate (75 ml). Then an ethyl acetate solution is then washed with a mixture of water/salt solution (70 ml), and the other with a mixture of water/saturated salt solution (60 ml), 1N sodium hydroxide (60 ml) and a third mixture of water/saturated salt solution (60 ml). The fortress of saturated salt solution was 12% of the mass. Then an ethyl acetate solution was dried with sodium sulfate, filtered and concentrated to dryness on a rotary evaporator, resulting in a received 4.5 g of a mixture of desired product and an unknown impurity. The ratio of impurity/product determined by HPLC-square, was about 50/15, respectively. The predominant product of this reaction was a mixture, which was identified as 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone.

The mixture was purified by column chromatography and obtained 1.9 g of analytically pure 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21-in primary forms, γ-lactone.

The residue was again analyzed and purified using chromatography. Conditions for HPLC: column Waters Symmetry C18 (150 mm × 4.6 mm votem.; the size of particles 5 microns); temperature column - room; mobile phase - acetonitrile/water 30/70 by volume; the pause flow 1.0 ml/min; volume of injection : 20 microlitres; the concentration of the sample - 1.0 mg/ml; UV detection at 210 nm; pressure - 1500 psi (105,5 kg/cm2); and the running time is 45 minutes. Conditions for TLC: adsorbent silica gel Merck 60 F254; the system solvent - chloroform/methyl tert-butyl ether/isopropanol, 70/28/2 volume; visualisation technique - 50% vol. water (H2SO4/LWUV and 50% vol. H2SO4/phosphomolybdenum acid; and the number of application - 100 micrograms.

Example 37i indigenous. Getting 7 Metelitza-17-hydroxy-3,11-dioxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone

The Jones reagent was prepared by dissolving 6.7 g of the anhydride of chromic acid (CrO3) in 6 ml of concentrated sulfuric acid and the 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 the secondary alcohol with obtaining ketone.

Complex hydroxyether (10.0 g, 24,0 mmol)obtained by the method described in Example 34 was dissolved/suspended in 1200 ml of acetone. To this mixture was added 8,992 ml of Jones reagent and the combined mixture was stirred for 10 minutes. An aliquot of the reaction mixture after treatment with water and extraction of Meiling what oricom analyzed by HPLC (column: Beckman Ultrasphere ODS C18, 4.6 mm × 250 mm, 5 μm; gradient solvent: acetonitrile/water=1/99-100/0 for 20 minutes at a flow rate of 1.5 ml/min; detector: UV 210 nm). On completion of the reaction was indicated by the absence of any significant amount of starting material in the reaction mixture. Retention time for the original material (complex hydroxyether) was 13,37 minutes, and for the product ketone - 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 again washed with 200 ml of water. The organic layer was separated from the aqueous layer and dried with magnesium sulfate. The solvent is evaporated to obtain 9,52 g of a whitish solid (yield crude substance is 95.6%) 7 Metelitza-17-hydroxy-3,11-dioxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone.

The structure was confirmed on the basis of the mass spectrum (m/e 414). H-NMR (DMF-d7) and C-NMR (DMF-d7). In the H NMR characteristic peak of the 11-N (4,51 ppm, doublet, J=5.8 Hz), found in complex hydroxyether, was absent. C-NMR was found to peak 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-Sooma), 167,21(C5), 125,33 (C4), 93,63(C17) and other peaks in the range of from 15 to 57 ppm

Example 37J. Getting dimethyl-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms is, γ-lactone

A solution of 3.5 g (8.4 mmol) of complex hydroxyether, obtained as described in Example 34, 42 ml of methanol was mixed with 4 ml of methanol 4n potassium hydroxide (8 mmol). The suspension was stirred over night at room temperature and heated under reflux for one hour. The methanol is evaporated in vacuo and the residue was mixed with 50 ml of ethyl acetate. The ethyl acetate evaporated in vacuo, and the residue is hydrolyzed in 50 ml of ethyl acetate. The dried solid was combined with 50 ml of acetone and 2 ml under the conditions (32.1 mmol). The mixture was stirred at room temperature for 18 hours. During this time a large part of the solids had dissolved. The mixture was filtered and the filtrate evaporated to dryness in a vacuum. The residue is hydrolyzed with ethyl acetate, after which the solids were removed by filtration and the solvent was removed by vacuum distillation. The residue was identified as a mixture of 78:22 (vol./about.) dimethyl-11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone and raw material complex hydroxyether, as defined using the1H-NMR. This mixture was suitable for use as an HPLC-token without additional purification.

1H-NMR (CDCl3) showed the following characteristic spectrum of 0.93 ppm(s, 3H), of 1.37(s, 3H), of 3.64(s, 3H), 3,69(who, 3H).

Example C. Receipt 11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone

To up 11,86 g (28.5 mmol) of complex hydroxyether obtained by the method described in Example 34, was added 50 ml of methanol and 20 ml of 2.5m NaOH. The suspension was heated under reflux. After 25 minutes, a portion of the original complex of ether, which, as shown by HPLC, remained unreacted (Bond SB-C8 150 × 4.6 mm, 2 ml/min, linear gradient of 35:65:45:55 And:within 15 minutes, A=acetonitrile/methanol 1:1, A=water/0.1% of triperoxonane acid, detection at 210 nm) and added 10 ml of 10M NaOH. After 1.5 hours, as shown by HPLC, there was only trace amounts of unreacted ether complex. The reaction mixture was left for about 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 obtained resinous residue was stirred up until the residue became a suspension. The solids were isolated by filtration, resuspendable in methanol and filtered, resulting in a received 3.75 g of brown solid. This substance was dissolved in 8 ml of hot DMF and the mixture was diluted with 40 ml of methanol. Acid was led and was isolated by filtration, which was obtained 1.7 g chapeaurouge white is solid substances, 11α,17-dihydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone.

1H-NMR (400 MHz, dateregistered) δ to 0.80(s, 3H), 1,25 (s, 3H), 1,2-2,7(m, 20N), 3,8(Sirs, 1H), 4,45(m, 1H), 5,50(s, 1H). Carboxyl proton was not observed due to the presence of HOD peak at 3,4 ppm

Example 38.

Scheme 1: stage 3C: Method G: Obtain 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone

Repeating the procedure of Example 37A except that, due to the treatment of the reaction solution ionoobmennoi resin, basic alumina or primary silicon dioxide, multiple stages of washing was not performed. Conditions for the processing of basic alumina or primary silicon dioxide described in Table Alo found that each of these treatments was effective to remove impurities without multiple stages of leaching of Example 44, resulting in a received 7 Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone.

Table 38.
FactorThe selected numberThe purpose of the experimentKey results
The main oxide of alumia2 g/125 g of productProcessing of the reaction mixture basic oxide is Yu aluminum removal Et 3N.HCl-salt, and to remove 1N NaOH and 1N HCl leachingThe yield was 93%
The main silicon dioxide2 g/125 g of productProcessing of the reaction mixture basic silicon dioxide, which is cheaper to remove Et3N.HCl-salt, and to remove 1N NaOH and 1N HCl leachingThe yield was 95%

Example 39.

Scheme 1: stage 3C: Method N: Obtain 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone

The potassium formate (4 g) and triperoxonane acid (42,5 ml) was mixed in a 100 ml reactor. Then to the mixture at a controlled rate added triperoxonane anhydride (9.5 ml), maintaining during the addition the temperature below 30°C. Then the solution was heated to 30°C for 30 minutes and cooled to room temperature, resulting in a received eliminating reagent used for the conversion of nelfinavir formula IV in complex enefer formula II.

Prior eliminating the anhydride reagent TFA/TFA was added to a solution of nelfinavir formula IV, previously obtained by the method described in Example 37A. The resulting mixture was heated to 40°C for 4.5 hours, and the degree of reaction was periodically monitored by TLC or HPLC. At the completion of the reaction, the mixture was transferred into a 1-neck is Yu flask and concentrated to dryness under reduced pressure and at room temperature (22° C). To the mixture was added ethyl acetate (137 ml), the resulting solid material was completely dissolved, and then added a mixture of water/saturated salt solution (137 ml) and the resulting biphasic mixture was stirred for 10 minutes. Then the mixture was left for 20 minutes to separate the phases. The fortress of saturated salt solution was 24% of the mass. The aqueous phase was subjected to contact with additional ethyl acetate (68 ml) and the thus obtained two-phase mixture was stirred for 10 min, after which it was left for 15 minutes to separate the phases. An ethyl acetate layers after two extraction were combined and washed for 24% of the mass. saturated salt solution (120 ml), another aliquot of 24% of the mass. saturated brine (60 ml), 1N solution of sodium hydroxide (150 ml) and one portion of saturated brine (60 ml). After each addition of the aqueous phase and 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°using the device for pumping water. The solid product (of 8.09 g) was analyzed by HPLC, as a result it was found that this product consists of 83.4% (square) complex tafira 7-methyl-hydro-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone; 2,45% (square is) 11,12-olefin; 1.5% of the 7,9-lactone; and 1.1% of unreacted nelfinavir.

Example 40.

Scheme 1: stage 3C: Method I: Getting 7 Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone

Mesilate, having a structure obtained in Example 23 (1.0 g), isopropenylacetate(10 g) and p-toluensulfonate acid (5 mg) were placed in 50 ml flask and heated to 90°while stirring. After 5 hours the mixture was cooled to 25°and concentrated in vacuum at 10 mm Hg, the Residue was dissolved in CH2Cl2(20 ml) and washed with 5% aqueous NaHCO3. CH2Cl2layer was concentrated in vacuum and received 1.47 g of yellow-brown oil. This product was recrystallized from CH2Cl2/Et2O and obtained 0.50 g of enolacetate formula IV(Z).

This product was added to a mixture of sodium acetate (0.12 g) and acetic acid (2.0 ml), which was pre-heated to 100°while stirring. After 60 minutes the mixture was cooled to 25°and diluted With CH2Cl2(20 ml). The solution was washed with water (20 ml) and dried MgSO4. The drying agent was removed by filtration and the filtrate was concentrated in vacuo, resulting in a received 0.4 g of the desired 9,11-olefin, 7 Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone. The crude product contained less than 2% impurities 7,9-lactone.

Example 41. Thermal eliminer is of nelfinavir in DMSO

Scheme 1: stage 3C: Method J: Obtain 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone

A mixture of 2 g of nelfinavir and 5 ml of DMSO in the reaction flask was heated at 80°With over 22.4 hours. HPLC analysis of the reaction mixture did not detect the source of the substance. To the reaction mixture were added water (10 ml) and the precipitate three times were extracted with methylene chloride. United methylenchloride layers were washed with water, dried with magnesium sulfate and concentrated to obtain complex tafira, 7 Metelitza-17-hydroxy-3-oxo-17α-pregna-4,9(11)-diene-7,21-in primary forms, γ-lactone.

Example 42.

Scheme 1: stage 3D: Method b: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

In a 50-ml flask pear shaped complex enefer formula IIA (1.07 g analyzed 74,4% complex enefer), trichloroacetamide (0.32 g) and decalibrated (0,70 g) in the form of a solid substance was mixed, while stirring with methylene chloride (15.0 ml). Then pipette for 1 minute was added hydrogen peroxide (30 wt%; 5.0 ml). The resulting mixture was stirred 6 hours at room temperature, during which time HPLC analysis showed that the ratio epoxyoctane to complex anaphero in the reaction mixture was approximately 1:1. In the reaction mixture introduced is more trichloroacetamide (0.32 g) and the reaction continued, stirring occasionally, for 8 hours, whereupon it was found that the residual number of complex tafira decreased to 10%. Then added more trichloroacetamide (0.08 g) and the reaction mixture was left overnight, after which the mixture was left only 5% of unreacted complex tafira towards epoxyoctane.

Example 43.

Scheme 1: stage 3D: Method C: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

In a 100 ml reactor were added complex enefer formula IIA (5,4 g, in accordance with the quantitative analysis of 74.4% complex enefer). To this complex anaphero added trichloroacetamide (4.9 g) and decalibrated (3,9 g), both in solid form and then added methylene chloride (50 ml). The mixture was cooled to 15°and within 10 minutes was added 30% hydrogen peroxide (25 g). The reaction mixture was left until then, until it was heated up to 20°and at this temperature, was stirred for 6 hours, after which it was performed HPLC analysis on the degree of transformation. It was found that the number of remaining tafira was less than 1% of the mass.

This reaction mixture was added to water (100 ml) and the mixture was left for phase separation, after which methylenchloride layer was removed. To methylenchloride layer was added sodium hydroxide (0,5h, 50 ml). After 20 minutes the mixture is left for phase separation and then to methylenchloride layer was added HCl (0,5h; 50 ml), after which the mixture was left for phase separation and the organic phase was washed with saturated saline (50 ml). Methylenchloride layer was dried with magnesium sulfate and the solvent was removed. The result obtained white solid (5.7 g). The aqueous layer aqueous sodium hydroxide solution was acidified and extracted, and the extract was treated with obtaining another 0.2 g of product. Output epoxyoctane was 90.2%.

Example 44.

Scheme 1: stage 3D: Method D: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Complex enefer formula IIA turned in epoxyoctane the method described in Example 43, except for the following changes: the initial download contains complex enefer (5,4 g, quantitative analysis: 74,4% complex enefer), trichloroacetamide (3.3 grams) and decalibrated (3.5 g). Then added hydrogen peroxide (12.5 ml). The reaction was carried out overnight at 20°after which, as shown by HPLC, was observed in 90%conversion of complex tafira in epoxyoctane. Then added 3.3 g of trichloroacetamide and 30% hydrogen peroxide (5.0 ml) and the reaction was conducted for 6 hours, after which the amount of residual complex tafira accounted for only 2% of the load calculation of complex enefer. After processing, a description is about in Example 43, received 5,71 g epoxyoctane.

Example 45.

Scheme 1: stage 3D: Method E: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Complex enefer formula IIA turned in epoxyoctane the method described in Example 43. In the reaction of this example, the loading complex tafira amounted to 5.4 g (quantitative analysis: 74,4% complex enefer), download trichloroacetamide was 4.9 g, loading of hydrogen peroxide was 25 g, and download dailybeast was 3.5, the Reaction was carried out for 18 hours at 20°C. the Amount of residual complex tafira was less than 2%. After processing the received 5,71 g epoxyoctane.

Example 45.

Scheme 1: stage 3D: Method F: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Complex enefer formula IIA turned in epoxyoctane the method described in Example 43, except that the reaction temperature of this sample was 28°C. Loading the reactor complex enefer (2.7 g), trichloroacetamide (2.5 g) and decalibrated (1.7 g), peroxide vodopada (17.0 g) and methylene chloride (50 ml). After 4 hours, the amount of residual complex tafira accounted for only 2% of the load calculation of complex enefer. After processing as described in Example 43, received 3.0 g epoxies is the northward.

Example 47-1.

Scheme 1: stage 3D: Method G: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Complex enefer formula IIA (40 g, quantitative analysis: 68.4% of complex enefer) were loaded into a 1,000 ml reactor with jacket and was dissolved in 175 ml of methylene chloride. The solution was stirred as adding trichloroacetamide (22,3 g) and dailybeast (6.0 g) in the form of solids. This mixture was stirred at 400 rpm and the temperature brought up to 27°using bath with a constant temperature for the regulation of the circulation of the fluid through the jacket of the reactor. Then in 3-5 minutes was added hydrogen peroxide (72,8 ml analysis: 30%). After adding hydrogen peroxide and the mixture was stirred at 400 rpm and 27°C. HPLC analysis indicated that for 5 hours the reaction was complete (99%). Six hours were added for 72.8 ml of water. Aqueous hydrogen peroxide is separated and once was 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 the methylene chloride initiated by distillation at atmospheric pressure and placed in the vacuum. Thus was obtained a yellowish residue (48,7 g, analysis of 55.4%). This corresponded to a yield of 94.8%, obtained from the analysis conducted in the calculation of the molar is output.

Part (47,8 g) of the residue was combined with 498 ml of 3A ethanol (95% ethanol, denatured with 5% methanol). The resulting mixture was heated under reflux and 249 ml of distillate was removed at atmospheric pressure. The mixture was cooled to 25°and filtered. Rinse with 3A ethanol (53 ml) was used to facilitate transfer. The dried solid was 27.6 g (analysis: 87,0%), which corresponded to a yield of 91%. Part of the solids (27,0 g) was dissolved in 292 ml of methyl ethyl ketone by heating under reflux. The hot solution was filtered through a layer of Solkafloc (powdered cellulose) with other 48.6 ml of methyl ethyl ketone, used to facilitate transfer. 146 ml portion of methyl ethyl ketone was removed by distillation at atmospheric pressure. The solution was cooled to 50°and was stirred for one hour as the crystallization of the product. After one hour the mixture was cooled to 25°C. the Stirring was continued for one hour and the solid was filtered from 48.6 ml of methyl ethyl ketone, used as a rinse. The solid was dried to constant mass of 20.5 g, which was output 87,2% after recrystallization. The yield of the reaction and dedicated ethanol, methyl ethyl ketone were combined to obtain the full yield of 75%.

Methylethylketone mother solution was suitable for re-cycle CC is based methylenechloride solution of the subsequent reactions. The combined mixture of 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 epoxyoctane. One half of the solvent was removed by distillation at atmospheric pressure. After cooling to 25°the solid was filtered and dried. Solid dry substance was dissolved in 12 volumes of methyl ethyl ketone by heating under reflux. The hot solution was filtered through a layer of Solka Floc with 2 volumes of methyl ethyl ketone, was added as a rinse. The filtrate was concentrated by distillation 6 volumes of methyl ethyl ketone at atmospheric pressure. This solution was cooled to 50°and was stirred for one hour as the crystallization of the product. After one hour the mixture was cooled to 25°C. the Stirring was continued for another one hour and the solid product was filtered with 2 volumes of methyl ethyl ketone used as a rinse. The solid product was dried to a constant mass. The introduction of a stock solution of methyl ethyl ketone increased the total yield up to 80-85%.

This method proved to be particularly suitable for large-scale production because it helps maximize the performance and minimize the volumes of leaching and waste.

Example 47A.

Scheme 1: stage 3D: Method N: With ntes Metelitza-9,11α -epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone

Complex enefer formula IIA (17 g, quantitative analysis: 72% complex enefer) was dissolved in methylene chloride (150 ml), then added trichloroacetamide (14.9 g) with slow stirring. The temperature of the mixture was brought to 25°and in the solution anafinova substrate under stirring at a speed of 400 rpm solution was added dailybeast (10.6 g) in water (10,6 ml). Then to the mixture of substrate/phosphate/trichloroacetamide in 3-5 minutes was added hydrogen peroxide (30% wt. solution; 69,4 ml). Exothermic reaction or oxygen evolution was observed. Thus obtained reaction mixture was stirred at 400 rpm in a period of 18.5 hours at 25°C. In the reaction, any oxygen evolution was not observed, but the analysis on consumption of hydrogen peroxide showed that during the reaction formed a certain amount of oxygen. 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 the temperature control was not necessary and the reaction was carried out mainly at room temperature (suitable is any temperature in the range of 5-25°). Aqueous and organic layers were left to separate and the lower methylenchloride layer was removed.

The water layer is, for example, the Ali reverse extraction with methylene chloride (69,4 ml) for 15 min, stirring at 250 rpm/min, the layers were left to separate and the lower methylenchloride layer was removed. The aqueous layer (177 g; pH=7) were subjected to analysis of hydrogen peroxide. The result (12,2%) analysis indicated that the reaction 0,0307 mol of olefin absorbed 0,0434 mol of hydrogen peroxide. Excessive absorption of hydrogen peroxide is an indicator of the formation of oxygen in this reaction. Back extraction of a small amount of methylene chloride was sufficient guarantee no loss of epoxyoctane in the water layer. This result coincides with the result obtained using the second extraction of a large number of methylene chloride, which was allocated only trichloroacetamide.

United methylenchloride solutions obtained from the above extraction was combined and washed with 3% of the mass. the sodium sulphate solution (122 ml) for at least 15 minutes under stirring at a speed of about 250 rpm At the end of the mixing period was obtained a negative test with the use of iodide of starch-KI paper; coloring was not observed; in a positive test is purple color indicated the presence of peroxide).

Aqueous and organic layers were left to their separation, and the lower methylenchloride layer was removed. The aqueous layer (pH=6) were discarded. It should be noted that the addition of a solution of sodium sulfite which may cause a small temperature increase, therefore, such addition should be carried out under temperature control.

Methylenchloride phase was washed with 0.5 n sodium hydroxide (61 ml) for 45 minutes at mixing speeds of about 250 rpm and at a temperature in the range of 15-25° (pH=12-13). During this operation, remove impurities originating from trichloroacetamide. Acidification of the alkaline aqueous fractions followed by extraction with methylene chloride confirmed that when this operation is lost very little epoxyoctane.

Methylenchloride phase once washed with 0.1 n hydrochloric acid (61 ml) for 15 minutes at a speed of stirring of 250 rpm and at a temperature in the range of 15-25°C. Then these layers were left to separate and the lower methylenchloride layer was removed and again washed with 10% mass. aqueous sodium chloride (61 ml) for 15 minutes when the stirring speed of 250 rpm and at a temperature in the range of 15-25°C. And the layers were again left to separate and the organic layer was removed. The organic layer was filtered through a layer of Solkafloc, and then was evaporated to dryness under reduced pressure. Drying was performed in a water bath at a temperature of 65°C. as a result received a whitish solid (17,95 g), which was subjected to HPLC analysis. The analysis showed that the yield epoxyoctane was 66,05%. Adjusted molar yield for this Rea is tion accounted for 93.1%of.

The product was dissolved in hot ethyl ketone (189 ml) and the resulting solution was subjected to distillation at atmospheric pressure until, until it was removed 95 ml of ketone solvent. The temperature was lowered to 50°as the crystallization of the product. Stirring was continued for 1 h at 50°C. Then the temperature was lowered to 20-25°and the stirring continued for another 2 hours. The solid was filtered, washed with MEK (24 ml) and the solid was dried to a constant mass 9,98 g, HPLC-analysis which indicated the content 93,63% epoxyoctane. 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 put another 18 ml of MEK as washing and filtered MEK solution was subjected to distillation at atmospheric pressure until, until it was removed 53 ml of solvent. The temperature was lowered to 50°as the crystallization of the product; and the stirring was continued at 50°C for 1 hour. Then the temperature was lowered to 20-25°and continued to stir at this temperature for another 2 hours. The solid product was filtered and washed with MEK (18 ml). Then the solid product was dried to a constant mass 8,32 g, which contained 99.6% of epoxyoctane, as it was shown by quantitative HPLC analysis. The final loss of the donkey drying was less than 1.0%. Total yield epoxyoctane, obtained by carrying out the reaction and treatment described in this Example was 65,8%. This total output was: the yield of the reaction is 93%, the allocation after the first crystallization 78,9% allocation after recrystallization 89,5%.

Example 47B. Getting 7 Metelitza-11α,12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone

Δ11,12-Olefin complex tafira is a by-product of the elimination of 11-nelfinavir. From the reaction mixture obtained by the method described in Example 37A, allocated net sample by repeated chromatography. Thus, 73 g of residue (obtained as described in Example 37A) was chromatographically 2.41 kg of Merck silica gel (40-63 μm) according to the scheme elution with a gradient of ethyl acetate, toluene(20:80, 30:70, 40:60, 60:40 about./vol.). Enriched Δ11,12the olefin part of the combined selected from 30:70 fractions. Selection of appropriate fractions were performed on the basis of TLC on EMF plates using ethyl acetate/toluene (60:40./about.) and visualization of sulfuric acid SWUV. 7.9 g of crude Δ11,12-olefin (80% by HPLC area)obtained after removal of solvent, was chromatographically on 531 g of Merck silica gel (40-63 μm) according to the scheme elution with a gradient of utilizationauthority (10:90, 20:80, 35:65./vol.). Pure 7-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,11-Dien-7α,21, in primary forms, γ-lactone (and 3.72 g) was obtained from the 20:80 fractions. The choice of the fractions was performed on the basis of TLC-evaluation, as described above.

MICK cm-11767 (lactone), 1727 (ester), 1668 and 1616 (3-keto-Δ4,5).

1H-NMR (CDCl3) of 1.05 ppm(s, 3H)and 1.15(s, 3H), 3,66(s, 3H), to 5.58(DD,1H), 5,80 (s, 1H), 5,88(DD, 1H).

13C-NMR (CDCl3) 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-Metelitza-17-hydroxy-3-oxo-17α-pregna-4,11-Dien-7α,21-in primary forms, γ-lactone in 16 ml of methylene chloride was mixed with 2.2 ml of trichloroacetonitrile (of 22.4 mmol) and 0.75 g of discalificata (4.3 mmol). This mixture was stirred and combined from 6.7 ml of 30% hydrogen peroxide (66 mmol). Stirring is continued at 25°C for 45 hours. After this time was added 28 ml of methylene chloride and 39 ml of water. The organic portion was isolated and washed sequentially: (a) 74 ml of 3% sodium sulfate, (b) 62 ml of 1N sodium hydroxide, (C) and 74 ml of 1N hydrochloric acid and (d) 31 ml of 10% saturated salt solution. The organic portion was again separated, dried with magnesium sulfate and evaporated to dryness in vacuum. 1,25 g of residue were chromatographically on 138,2 g of Merck silica gel (40-63 μm) using a gradient of methyl tert-butyl ether and is of awala (40:60, 60:40, 75:25./vol.). The relevant portions 60:40, 75:25 fractions were combined based on TLC-assessment, and received 0.66 g of pure 7-methyl-hydro-11α,12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone. In the TLC system used EMF plate circuit elution of methyl tert-butyl ether and toluene 75:25 (vol./about.) and using sulfuric acid and SWUV for rendering.

1H-NMR (CDCl3) ppm of 1.09(s, 3H), of 1.30(s, 3H), 3,05(AB11,12to 2N), to 3.67(s, 3H), 5,80(s, 1H).

13C-NMR (CDCl3) 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: 69,54 and N 7,30; Found 69,29 and N 7,17.

Example 47S. Selection 7 Metelitza-4α,5α:9α,11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α,21-in primary forms, γ-lactone

Untreated epoxyoctane (157 g), obtained from 200 g of complex tafira the method described in Example 26, was subjected to chromatography on a 4.5 kg of Merck silica gel (40-63 μm). at 88.1 g portion was allocated using the scheme elution of acetonitrile and toluene 10:90 (vol./vol.). Selected solid was dissolved in 880 ml of hot methyl ethyl ketone and filtered through a layer of Solka Floc. Then put another 88 ml of methyl ethyl ketone as a rinse. The filtrate was concentrated by removing 643 ml of solvent, and the mixture was cooled whom to room temperature. The solids were filtered and washed with methyl ethyl ketone. After drying, received a 60.2 g epoxyoctane that amounted to 96.8% according to HPLC. The filtrate was concentrated to dryness under reduced pressure. 9.3 g of the residue was recrystallized from 99 ml of methyl ethyl ketone and received 2.4 g dry solids. Portion 400 mg of this solid was subjected to reverse-phase preparative HPLC on a column of YMC ODS AQ. Pure 7-methyl - hydro-4α,5α:9α,11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α,21, in primary forms, γ-lactone (103 mg) was isolated using the scheme elution of acetonitrile (24%), methanol (4%) and water (72%).

1H-NMR (CDCl3) ppm and 0.98(s, 3H), 1,32(s, 3H), 2,89(m, 1H), 3,07(C,d, 2H), of 3.73(s, 3H).

MS, M+430, calculated for C24H30O7(430,50).

Example 47D. Selection 7 Metelitza-17-hydroxy-3,12-dioxo-17α-pregna-4,9(11)-Dien-7α,21-in primary forms, γ-lactone

Uterine methylethylketone solution obtained by the method described in Example 26, was evaporated to dryness under reduced pressure. 4.4 g portion of the residue was subjected to chromatography on 58,4 g BTR Bond LP (40 μm). After elution gradient of ethyl ketone and methylene chloride (25:75-50:50./about.) got to 1.38 g of product. a 1.3 g portion of this product was further purified using reverse-phase preparative who HPLC using as mobile phase of acetonitrile (30%), methanol (5%) and water (65%) and column YMC ODS AQ (10 μm). This product was obtained from enriched fractions by extraction with methylene chloride. The methylene chloride evaporated to dryness and 175 mg of the residue was purified again by reverse-phase preparative HPLC using a mobile phase of acetonitrile (24%), methanol (4%) and water (72%) and column YMC ODS AQ. Extraction of enriched fractions of methylene chloride gave 30,6 mg of pure 7-Metelitza-17-hydroxy-3,12-dioxo-17α-pregna-4,9(11)-Dien-7α,21-in primary forms, γ-lactone.

1H-NMR (CDCl3) ppm of 1.17(s, 3H), 1,49(s, 3H), of 3.13(m, 1H), 3,62(s, 3H), 5,77(s, 1H), 5,96 (s, 1H).

13C-NMR (CDCl3) 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 E. Getting 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid, dihydrate, Pikalevo salt

Received a suspension containing 2.0 g (4.8 mmol) epoxyoctane, obtained as described in Example 43, 10 ml 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. during the first two hours were formed yellow homogeneous solution. The temperature was raised to 70°and the stirring was continued for further 3 hours. The solvent was removed by vacuum distillation and the residue was purified using about Ameno-phase HPLC on 90 g of silica gel C18 using water as eluent. The desired fractions were combined according to the evaluation carried out 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 subjected to purification using reversed-phase HPLC performed as described above. The desired fractions were concentrated to dryness under reduced pressure, and the residue was dissolved in ethanol. For the determination of cloud temperature was added ethyl acetate, and then was added heptane to complete the deposition. Was allocated 0.55 g of product 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid, dihydrate, Pikalevo salt, as a yellow solid. Carbon analysis was consistent with the hydrated structure With23H28O7K2.1,75 H2O: Calculated C, 52,50 as compared with 55,85 for hydrated forms; found 52,49. After carrying out TLC on EMF plates using methylene chloride, methanol and water (6:3:0.5 to about./about.) as eluent and visualization using SWUV observed Rf=0,29.

Example 47F. Getting 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid, disodium salt

About 5 mg (0.01 mmol) epoxyoctane obtained by the method described in Primera, suspended in 200 µl of methanol (4 ml vessel and diluted to approximately 200 ál of 2.5 NaOH. The resulting mixture had a yellow color and was homogeneous. Then the mixture was heated in an oil bath at 70°C. After 10 minutes, 1 μm of the sample taken from this mixture was analyzed by HPLC (Bond SB-C8, 150 × 4.6 mm, 2 ml/minute, gradient = 35:65 (vol./about.) A: b, a = acetonitrile/methanol (1:1), A=water/0.1% of triperoxonane acid, detection at 210 nm), and this analysis revealed two products with retention times of 4,86 and 2.93 minutes, which corresponded to the hydroxy acid (non-closed lactone) and 7-carboxylic acid with closed lactone, respectively. After 30 minutes, take a second sample (0.05 ml) and acidified 0.05 ml of 3n HCl and then neutralized with approximately 0.5 ml of sodium bicarbonate. HPLC-analysis, as above, was found expected steroids with sanothimi rings at the retention time 6,59 and 10,71 minutes. Attitude 7 Metelitza-9,11α-epoxy-17-hydroxy-3 - oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone (10,71 min) to the corresponding 7-carboxylic acid was 7:89.

Selective hydrolysis of the lactone can be performed under mild conditions. The second 4 ml vessel was prepared as described above, but without heating. The mixture was treated with ultrasound for 5 minutes. 0.05 ml of sample was diluted in 0.5 ml of a mixture of methanol/acetonitrile, 1:1 (vol./collection), and analyzed with the aid of the d HPLC without prior acidification. The obtained complex 7-ether carboxylic acid with an open the lactone had a retention time 4,85 minutes, as was observed above, and had no impurities in the form of 7-carboxylic acid.

Example 47G. Selection 7 Metelitza-9α,11β17th trihydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone

and 7 Metelitza-12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21-in primary forms, γ-lactone

7 Metelitza-9α,11β17th trihydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone and 7-Metelitza-12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21, in primary forms, γ-lactone was isolated after carrying out preparative liquid chromatography 2-betononasos the mother liquor obtained in the epoxidation of complex tafira as described in Example 26 (scheme using trichloroacetonitrile). For this first crystallization was performed using the specified 2-butanone. However, for the recrystallization instead of acetone used 2-butanone (10 capacities per 1 g). This method was obtained 2.8 g of residue, which was purified using reverse-phase preparative HPLC. As stationary phase used Cromasil C8 (10 μm)as the mobile phase consisted of milliQ water and acetonitrile in the ratio of 70:30 (vol./vol.). Cree is talisay was observed in one of the enriched fractions. Solid (46,7 mg) was isolated and identified as 7-Metelitza-9α,11β17th trihydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone. The mother liquor was evaporated to dryness under reduced pressure and the residue (123 mg) was identified as 7-Metelitza-12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21, in primary forms, γ-lactone. 7 Metelitza-9α,11β17th trihydroxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone:

MS M+432 calculated for C24H32O5(432,51).

1H-NMR (CDCl3) of 1.23 ppm(s, 3H), and 1.54(s, 3H), of 3.00(m, 1H), 3,14(m, 1H), 3,74(s, 3H), 5,14(s, 1H, slowly exchanged), 5,79 (s, 1H).

13C-NMR (CDCl3) 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 Metelitza-12α,17-dihydroxy-3-oxo-17α-pregna-4,9(11)-Dien-7α,21, in primary forms, γ-lactone:

MS M+441 calculated for C24H30About5(414,50).

1H-NMR (CDCl3) of 0.87 ppm(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).

13C-NMR (CDCl3) 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 N. Getting 7 Metelitza-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone

and 7 Metelitza-6β,17-dihydroxy-9,11αEpoque and-3-oxo-17α -pregn-4-ene-7α,21-in primary forms, γ-lactone

7 Metelitza-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone was obtained by the method described in R.M.Weier & L.M.Hofmann (J.Med.Chem. 1977, 1304), which is introduced in the present description by reference. 148 g (357 mmol) of 7-Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone, obtained by the method described in Example 43, combined with 311 ml of absolute ethanol and 155 ml (932 mmol) triethylorthoformate. The suspension was stirred at room temperature and the catalyst was added 10.4 g (54,7 mmol) toluensulfonate acid (monohydrate). Stirring was continued for 30 minutes and the reaction was suppressed by adding to 41.4 g (505 mmol) of powdered sodium acetate and 20.7 ml (256 mmol) of pyridine. Solids (70,2 g) was removed by filtration and the filtrate was concentrated to dryness in vacuo. The residue is 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 hydrolyzed in 100 ml of methanol containing 2 ml of pyridine. and 29.7 g of solids were removed by filtration. The filtrate was observed additional deposition. Therefore, the filtrate was re-filtered to remove more of 21.9 g of solid substances the TV. The obtained filtrate was concentrated to dryness and the residue hydrolyzed in 50 ml of methanol containing 1 ml of pyridine. of 33.8 g of solids were isolated by filtration. Qualitative HPLC analysis indicated that this last portion of the solids was clean enough (90% in area 7 Metelitza-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone) for use in a subsequent reaction stage.

7 Metelitza-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone:

1H-NMR (CDCl3) ppm: 1,02(s, 3H), of 1.27(s, 3H), of 1.30(t, 3H), of 3.12(m, 1H), or 3.28(m, 1H), 3,66 (s, 3H), of 3.78(m, 2H), 5,20 (s, 1H), from 5.29(d, 1H).

8 g portion of the enol ether (7 Metelitza-9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone) (18 mmol)obtained in the previous stage was dissolved in 120 ml of 1,4-dioxane. This solution was combined with a mixture of 6.8 g of 53% m-chloroperoxybenzoic acid (to 20.9 mmol), and 18.5 ml of 1,0N sodium hydroxide (18.5 mmol) and 46 ml of dioxane/water (9:1). The temperature was maintained at -3°and the mixture was stirred for two hours. Then the temperature was raised to 25°and the stirring continued for another 20 hours. The mixture was combined with 400 ml of cold water (10° (C) and 23.5 ml of 1,0N sodium hydroxide (23.5 mmol). The mixture was extracted four times with 100 ml portions of methylene chloride (every time). Volume of the United methylenchloride portion was dried with magnesium sulfate and the solvent of the supernatant liquid was removed by distillation in vacuum. of 13.9 g of the residue is triturated with 50 ml of ethyl ether to obtain 2.9 g of white solid. 2.4 g portion of the solid was chromatographically 100 g of Merck silica gel (60 microns). After the initial rinse 1 liter of ethyl acetate/heptane (1:1), the product was suirable with ethyl acetate/heptane 7:3. Enriched fractions were combined based on TLC-assessment (plates with EMF; eluent: ethyl acetate/heptane (7:3 V/V); visualization using SWUV). Thus was obtained 0.85 grams of enriched substances and recrystallized it from 10 ml of isopropanol to obtain 0.7 g of 7-Metelitza-6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone. Fractions with a large number of impurities were combined and obtained 0.87 g of the crude 7-Metelitza-6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone. This product has chromatographically by 67.8 g of Merck silica gel (40-63 μm). Even 0,69 g of product were isolated by using toluene containing 0.5-2.5% methanol. 7 Metelitza-6β,17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregn-4-ene-7α,21, in primary forms, γ-lactone:

Calculated: 66,96 and N 7,02: Found: 66,68 and N 7,16.

1H-NMR (CDCl3): ppm: 1,06(s, 3H), 1,36(DM, 1H), and 1.63(s, 3H), of 2.92(m, 1H), to 3.02(DD, 1H), 3,12(d, 1H), to 3.64(s, 3H), br4.61(d, 1H), 5,96 (s, 1H).

13C-NMR (CDCl3) ppm: 16,17; 21,32; 21,79; 24,36; 27,99; 28,94; 30,86; 31,09; 32,75; 33,19; 34,92; 3,77; 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. Receiving 7 Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone

To 2 g (4.8 mmol) of 7-Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone, obtained by the method described in Example 43 was added 3.3 ml (14.4 mmol) of 25% sodium methoxide in methanol. The obtained yellow suspension was heated to 50°C. the Solid did not dissolve. To the mixture was added 3.3 ml of methanol (anhydrous methanol, Aldrich). Then the mixture was heated under reflux (65°) and this mixture became homogeneous. After 30 minutes, the solid precipitate was preventing the mixing.

Then add about 25 ml of anhydrous methanol and the mixture was transferred into a 100 ml flask. This mixture was heated under reflux for 16 hours, during which it became dark and homogeneous. The mixture was cooled to 25°and added 70 ml of 3n HCl (exothermic reaction). For cooling of the mixture were added a few grams of ice and the solution was extracted with two successive 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). Silicon dioxide was suirable 100 ml of methyl is of chloride. Then suirvey methylene chloride was concentrated in vacuum and received 1 g of a brown foam, which crystallized after addition of ethyl acetate. A layer of silicon dioxide was suirable second time with 100 ml of 10% ethyl acetate/methylene chloride and suirvey the solution was concentrated, resulting in a received 650 mg of a brown foam.

Thin layer chromatography (E.Merck, 60 F-254, 0.25 mm silica gel, toluene/ethyl acetate (1:1 vol./about.)) revealed the presence of 7-Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone and 7-Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7β,21-in primary forms, γ-lactone in both samples, although in the first sample was attended by a very small amount of 7α-carboxy-epimer. The first sample triturated with hot ethyl acetate (77° (C) and left to cool to 25°C. the mixture is Then filtered to obtain 400 mg whitish solids, TPL 254-258°C. H,13C and13With ART confirmed the established structure. A small amount of ethyl acetate remained in the sample, but the presence of the parent compound was not detected by HPLC (Bond SB-C8, 150 × 4.6 mm, 2 ml/minute, socrata mixture =40:60 (vol./about.) A:b, a = acetonitrile/methanol (1:1), A = water/0.1% of triperoxonane acid, detection at 210 nm) (HPLC showed 98.6% of the area) and TLC (toluene-ethyl acetate, 1:1,vol/vol.).

FAB-MS confirmed the molecular weight of 414 M+N when 415,2.

1H-NMR (400 MHz, deuterochloroform) δ of 0.95(s, 3H), 1,50(s, 3H), of 1.45(m, 3H), 1.55V is 2.7(m, 15 NM), 2,85(t, J=13, 1H), 3,25(d, J=6, 1H), the 3.65(s, 3H), 5,78(s, 1H).

Example 47J. Getting 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-dicarboxylic acid γ-lactone

To 774 mg (1.82 mmol) of 7-Metelitza-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α,21-in primary forms, γ-lactone, obtained by the method described in Example 43, and suspended in 3 ml of acetonitrile, was added 3 ml (7.5 mmol, 2.0 equivalent) 2,5M sodium hydroxide. The mixture became yellow, and after 10 minutes it became homogeneous.

To track the progress of reaction aliquots (0.1 ml) the mixture was suppressed in 0.01 ml of 3M sulfuric acid and was extracted in a glass 4 ml vessel with ethyl acetate (0.2 ml). The separation of the phases was carried out by removing the lower aqueous phase with a pipette. The organic phase was evaporated, and the residue was analyzed using the HPLC method described in Example N. After 50 minutes at 25°there were slight changes in the composition of the mixture.

The mixture was heated under reflux (about 90° (C) for 50 minutes. HPLC analysis of the mixture indicated the presence of 6% of the area of the remaining source material. The mixture was stirred for 65 hours at 25°C. Acidification, extraction and HPLC-Ana is from aliquots, carried out as described above, confirmed the absence of the remaining source material.

The mixture was strongly acidified by adding about 4 ml of 3M sulfuric acid and was extracted with two portions (about 10 ml) of methylene chloride. The organic phases were combined and dried with sodium sulfate. After concentration on a rotary evaporator received 780 g of solid substance. This solid is recrystallized from dimethylformamide/methanol to obtain 503 mg (67%) of yellowish-brown crystalline solid. With the rapid heating of the sample was melted with a gas at a temperature close to 260°C. slow heating to 285°the sample was slowly getting dark, but still solid.

1H-NMR (dimethylsulfoxide-d-6, 400 MHz) δ of 0.85(s, 3H), 1,4(s, 3H), from 1.3 to 2.9(m, N)and 3.15(m, 1H), of 5.55(s, 1H), 11.8 in(lat., 1H).

Example C.

Scheme 1: stage 3D: Method I: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

0.2 M Solution of complex tafira formula IIA in methylene chloride was combined with 2 equivalents of potassium phosphate, dissolved in an equal weight of water (50% wt./mass. aqueous solution), 3 equivalents of chlortetracycline and 22 equivalents of hydrogen peroxide (added as a 30% aqueous solution). Then the mixture was stirred at 25°C for 23 hours. The reaction specieslevel amount of water, equal loading of hydrogen peroxide, and the methylene chloride was separated. Methylenchloride part once washed with 3% solution of sodium sulfite (with volume equal to 1.75 times the loading of hydrogen peroxide). Methylenchloride part was separated and dried with sodium sulfate. The solution was concentrated by distillation at atmospheric pressure until such time as the temperature in the head part has not reached 70°C. the Residue was estimated using HPLC,1The h and1C-NMR (CDCl3). It was determined that the output epoxyoctane was a 54.2%by area HPLC.

Example 47L.

Scheme 1: stage 3D: Method J: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

The method described in Example C was repeated, but instead of chlortetracycline used getattributename (CF3CF2CF2CONH2). Output epoxyoctane was 58.4% by area HPLC.

Example 48. Epoxidation of complex tafira formula IIA using toluene

Scheme 1: stage 3D: Method: Synthesis of Metelitza-9,11α-epoxy-17α-hydroxy-3-oxoprop-4-ene-7α,21-in primary forms, γ-lactone.

Complex enefer formula IIA turned in epoxyoctane way, mainly described in Example 46, except that as a solvent was used toluene. The reactor was loaded such substances, ka is difficult enefer (2.7 g), trichloroacetamide (2.5 g), decalibrated (1.7 g), hydrogen peroxide (17.0 g) and toluene (50 ml). The reaction was left to raise the temperature to 28°and this reaction was completed after 4 hours. The obtained three-phase mixture was cooled to 15°C, filtered, washed with water and dried in vacuum to yield 2.5 g of product.

Example 49.

Figure 4: Method a: Epoxidation 9,11-dienone

The connection marked XVIIA (compound XVII, where both-And-And - and-In-To - represent-CH2-CH2-) (40,67 g), was dissolved in methylene chloride (250 ml) in one litre 3-necked flask, and then cooled from the outside with a mixture of ice and salt. Then was added potassium phosphate (22,5 g) and trichloroacetonitrile (83,5 g) and the mixture was cooled to 2°With, then within one hour was slowly added 30% hydrogen peroxide (200 g). The reaction mixture was stirred for 12°C for 8 hours and at room temperature for 14 hours. Then took a drop of the organic layer and was evaluated for the presence of any source of northward and found that it is <0.5 percent. After this was added water (400 ml), was stirred for 15 minutes and the layers were separated. The organic layer is successively washed with 200 ml of potassium iodide (10%), 200 ml of sodium thiosulfate (10%) and 100 ml saturated sodium bicarbonate solution, separating the layers each time. The organic layer was dried with anhydrous magnesium sulfate and concentrate who has demonstrated obtaining the crude epoxide (41 g). This product has led from ethyl acetate: methylene chloride to obtain 14.9 g of pure product.

Example 50.

Figure 4: Method: 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. and Then for 15 minutes, stirring, was added solid m-chloroperbenzoic acid (purity 50-60%; 21,86 g). Temperature increases were not observed. The reaction mixture was stirred for 3 hours and evaluated for the presence of dienone. This reaction mixture was sequentially treated with a solution of sodium sulfite (10%), sodium hydroxide solution (0.5 n), hydrochloric acid (5%) and, finally, 50 ml of saturated salt solution. After drying with anhydrous magnesium sulfate and evaporation got 17,64 g of epoxide and used directly in the next stage. It is estimated that this product contains a product of oxidation of Bayer-Villiger, which was removed by trituration with ethyl acetate, followed by crystallization from methylene chloride. Based on the scale of 500 g of precipitated m-chloroperbenzoic acid was filtered, followed by standard processing.

Example 51.

Figure 4: Method With: Epoxidation of compound XVIIA using trichloroacetamide

Compound XVIIA (2 g) was dissolved in 25 ml methylenchlorid the and. Then add trichloroacetamide (2 g) and dailypost (2 g). Stirring at room temperature, was added 30% praxis hydrogen (10 ml) and stirring continued for 18 hours to obtain epoxide (1.63 g). Product Bayer-Villiger not formed.

Example 52.

In a 2000 ml flask was loaded potassium hydroxide (56,39 g; 1005,03 mmol; 3.00 EQ.) and suspended with dimethylsulfoxide (750,0 ml) at room temperature. Then the flask with THF (956,0 ml) was loaded Trianon corresponding to formula XX (where R3is N, and each of the-a-a - and-B-B - represents-CH2-CH2-) (100,00 g; 335,01 mmol; 1.00 EQ.). After that the flask was loaded methyl sulfate trimethylsilane (to 126.14 g; 670,02 mmol; 2.00 EQ.) and the resulting mixture was heated under reflux for 1 h at 80-85°C. For the conversion of 17-spiroxamine was monitored by HPLC. Approximately 1 l of THF is evaporated from the reaction mixture under vacuum, and then within 30 minutes we were loaded water (460 ml) and the reaction mixture was cooled to 15°C. the resulting mixture was filtered and the solid oxiranyl product twice washed with 200 ml aliquot of water. As illustrated, the product was highly crystalline and perform its filtering was easy. Then the product was dried in vacuum at 40°C. has Been allocated 104,6 g simple 3-methyl enol ether is army Δ -5,6,9,11-17-oxiranemethanol product.

Example 53.

In a dry 500 ml reactor in a stream of nitrogen was loaded ethoxide sodium (41,94 g; 616,25 mmol; 1,90 EQ.). Then, the reactor was loaded ethanol (270,9 ml) and sodium methoxide suspended in ethanol. This suspension was loaded diethylmalonate (103,90 g; 648,68 mmol; 2.00 EQ.), then added oxiran-steroid, obtained as described in Example 52 (104,60 g; 324,34 mmol; 1.00 EQ.), and the resulting mixture was heated under reflux, that is, at 80-85°C. Heating was continued for 4 hours, after which the completion of the reaction was monitored by HPLC. In the reaction mixture within 30 minutes we were loaded water (337,86 ml) and the mixture was cooled to 15°C. the Stirring was continued for 30 minutes and then the reaction suspension was filtered to obtain the precipitate on the filter containing finely dispersed amorphous powder. The residue on the filter was washed twice with water (200 ml each)and then dried at room temperature under vacuum. Allocated 133,8 g 3-methyl-enlever-Δ-5,6,9,11-17-spirolactone-21-ethoxycarbonyl intermediate product.

Example 54.

3-Methyl-enlever-Δ-5,6,9,11-17-spirolactone-21-ethoxycarbonyl intermediate compound (formula XVIII, where R3is N, and each of the-a-a - and-B-B - represents-CH2-CH2-; 133,80 g; 313,68 mmol; 1.00 EQ., obtained as described in Example 53) instead of the ones with sodium chloride (of 27.50 g; 470,52 mmol; 1.50 EQ.), dimethylformamide (709 ml) and water (5 ml) were loaded with stirring in 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 for completion of the reaction. Then to the mixture for 30 minutes was added water and the water was cooled to 15°C. the Stirring was continued for 30 minutes, after which the reaction suspension was filtered, the result of which was allocated amorphous solid reaction product in the form of sediment on the filter. The residue on the filter twice washed (200 ml aliquot of water, and then dried. After drying, got to 91.6 g of the product, 3-melanolepis-17-spirolactone (output 82,3%; 96%of analysis area).

Example 55.

The enol ether 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) was loaded into a 2000 ml reactor, and the resulting suspension was heated under reflux for 2 hours. Then loaded water (600 ml) for 30 minutes and during this time the reaction mixture was cooled to 15°C. After the reaction, the suspension was filtered and the residue on the filter is twice washed with water (200 ml aliquot). 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 R3is N, and-And-And - and-In-what - are-CH 2-CH2-; output 95,9%).

Example 56.

Compound XVIIA (1 kg; 2,81 mol) were loaded with carbon tetrachloride (3.2 l) in a 4-necked 22-liter flask. To the mixture was added N-bromosuccinimide (538 g), and then acetonitrile (3.2 liters). The resulting mixture was heated under reflux and maintained at this temperature 68°C for approximately 3 hours, which formed the clear orange solution. After 5 minutes nagrania the solution became dark. After 6 hours the heating source was removed and samples were taken of the mixture. The solvent is evaporated in vacuo and to the residue at the bottom of the vessel was added ethyl acetate (6 l). The resulting mixture was stirred, after which was added 5%sodium bicarbonate solution (4 l) and the mixture was stirred for 15 min, after which it was left for phase separation. The aqueous layer was removed and the mixture was injected saturated salt solution (4 l), after which the mixture was stirred for 15 minutes Then the mixture is again left for phase separation and the organic layer was evaporated in vacuum to obtain a viscous suspension. Then was added dimethylformamide (4 l) and the evaporation was continued at the temperature of the vessel 55°C. the Residue at the bottom of the vessel was left overnight to ostivone, and then was added DABCO (330 g) and lithium bromide (243 g). Then the mixture was heated to 70°C. After half of the heat took samples of liquid chrome is adopted, after heating for 3,50 hours was added DABCO (40 g). After heating for 4.5 hours was injected water (4 l) and the resulting mixture was cooled to 15°C. the suspension was filtered and the filter cake washed with water (3 l) and dried on the filter overnight. Wet sediment (978 g) was loaded again in a 22 l flask was added dimethylformamide (7 l). Thus obtained mixture was heated to 105°C, after which the precipitate was completely dissolved in solution. The heating source was removed and the mixture in the flask was stirred and cooled. In the jacket of the reactor was poured into ice water and the mixture inside the reactor was cooled to 14°C and maintained for 2 hours. The resulting suspension was filtered and washed twice with 2.5 l aliquot of water. The filter cake was dried in vacuum over night. Received 510 g of light brown solid product.

Example 57.

A 2-liter, 4-necked flask was downloaded: 9,11-epoxiconazol, obtained as described in Example 56 (100,00 g; 282,1 mmol; 1.00 EQ.), dimethylformamide (650,0 ml), lithium chloride (30,00 g; 707,7 mmol; of 2.51 EQ.) and cyanohydrin acetone (72,04 g; 77,3 ml; 846,4 mmol; 3.00 EQ.). The resulting suspension was subjected to mechanical stirring and treated with tetramethylguanidine (45,49 g; 49,6 ml; 395,0 mmol; 1,40 EQ.). Then the system was filtered using a water cooled fridge and refrigerator with dry ice (filled with the chemical ice in acetone) to prevent leakage HCN. Exhaust pipe from the cooler with dry ice was added to the scrubber filled with a significant excess of chlorine bleach. This mixture was heated up to 80°C.

After 18 hours got dark reddish-brown solution, which was cooled to room temperature under stirring. In the process of cooling the solution was barbotirovany nitrogen to remove residual HCN by means of the exhaust pipe leading to the scrubber with bleach. After 2 hours the solution was treated with acetic acid (72 g) and was stirred for 30 minutes. Then the crude mixture was poured, with stirring, into ice water (2 l). After this mixed suspension was treated with 10% aqueous HCl (400 ml) and was stirred for 1 hour. Then the mixture was filtered to obtain a dark brick-red solid (73 g). The filtrate was placed in 4-liter separating funnel and was extracted with methylene chloride (3×800 ml); the organic layers were combined and subjected to back extraction with water (2×2 l). Methylenchloride the solution was concentrated in vacuum to obtain 61 g of a dark red oil.

After water wash fractions were left for the night, formed a considerable amount of sediment. This precipitate was collected by filtration and was determined that he is clean enaminones product (14.8 g).

After drying, the original red is solid substance (73 g) was analyzed by HPLC and were identified the main component is 9,11-epoxyresin. In addition, HPLC showed that the enamine was the main component of red oil obtained by treatment with methylene chloride. The calculated molar yield the enamine was 46%.

Example 58.

9,11-epoxyresin (4,600 g; 0,011261 mol; 1.00 EQ.), obtained as described in Example 57, was introduced in 1000 ml round bottom flask. To the mixture was added methanol (300 ml) and 0.5% of the mass. aqueous HCl (192 ml), after which the mixture was heated under reflux for 17 hours. Then the methanol was removed in vacuum, which resulted in a reduction in the number of substances in Cuba distilling up to 50 ml and to the formation of a white precipitate. To the suspension was added water (100 ml), and then it was filtered to obtain a white solid residue, which was three times washed with water. The output of the solid 9,11-amoxicillindosage product was 3,747 g (81,3%).

Example 59.

Epoxidation obtained by the method described in Example 58 (200 mg, 0.49 mmol), suspended in methanol (3 ml) and to the mixture was added 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU). After heating under reflux for 24 hours the mixture became homogeneous. It was then concentrated to dryness at 30°With a rotary evaporator and the residue was distributed between methylene chloride and 3,0N HCl. After concentrating the organic phase was awarded a yellow solid (193 mg), which, ka is determined, represented 22% of the mass. epoxyoctane. The yield was 20%.

Example 60.

To 100 mg of the diketone (obtained in accordance with Example 58), suspended in 1.5 ml of methanol, was added 10 microliters (0,18 EQ.) 25% (wt./mass.) solution of sodium methoxide in methanol. The solution was heated under reflux. After 30 minutes diketone were found, but were present complex 5-cyanoethyl. To the mixture was added 46 microlitres 25% (wt./mass.) methanol solution of sodium in methanol. The mixture was heated under reflux for 23 hours when using HPLC revealed that the main product is epoxyoctane.

Example 61.

To 2 g of the diketone (obtained in accordance with Example 58), suspended in 30 ml of dry methanol was added to 0.34 ml of triethylamine. 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 obtain 1.3 g of complex 5-canoeiro in the form of a white solid.

To 6.6 g of the diketone, suspended in 80 ml of methanol, was added to 2.8 ml of triethylamine. The mixture was heated under reflux for 4 hours and stirred at 25 rpm for 88 hours, during which the product crystallized from solution. As a result, the e filter and then washing with methanol, there was obtained 5.8 g of complex canoeiro in the form of a white powder. This powder was led from chloroform/methanol to obtain 3.1 g of crystalline product which was homogeneous, as evidenced by HPLC.

Based on the above we can say that we have achieved some of the objectives of the present invention and obtained other primary results.

It should be noted that although in the above described compositions and methods can be made various changes, not beyond the scope of the present invention, however, all options described in this application and illustrated in the accompanying drawings are illustrative and should not be construed as a limitation on the merits of the invention.

New connections

In addition, the present invention relates to additional polycyclic organic molecules, which can be used as chromatographic markers when receiving steroid compounds with predominant biological activity, such as spironolactone or epoxyoctane.

In short, it was found that some compounds containing substituted or unsubstituted steroid nucleus and substituted or unsubstituted carbocyclic ring is condensed with steroids the m kernel 13,17-position, can be used as internal or chromatographic markers when receiving steroids, such as spironolactone and epoxyoctane. In particular, the connection methyl-2,3,3A,4,6,7,9,10,11,11a,12,13-dodecahydro-3Aβ,11aβ-dimethyl-1,9-dioxo-1H-pentalen[1,6A-a]phenanthrene-6α-carboxylate:

can be used as chromatographic marker. One of the new distinctive characteristics of this compound and related compounds of the present invention is condensed carbocyclic ring attached to the D ring of the steroid nucleus. Spironolactone and epoxyoctane not have this characteristic, but instead they contain 20-spirolactone ring.

Mentioned in this description of the steroid core of this compound corresponds to the following structure:

This structure has the standard numbering and designations of the rings, taken to steroid compounds. Steroid nucleus may be saturated, unsaturated, substituted or unsubstituted. It contains, preferably, at least one to four unsaturated bonds. More preferably, when each of rings a, C and D contains at least one unsaturated bond. Steroid nucleus may also be substituted, as more specifically absurdes is below. This engine replaced, preferably at least C7-ester group.

Referred to in the present description carbocyclic ring is condensed with a steroid nucleus corresponds to the four-, five - or six-carbon cyclic skeleton. It can be saturated, unsaturated, substituted or unsubstituted. Preferably it is saturated or has one double bond and substituted hydroxy or keto-group. In addition, the carbocyclic ring preferably has a α-orientation in relation to the steroid nucleus.

In a preferred embodiment of the invention carbocyclic ring contains five-carbon cyclic skeleton and has α-orientation and a specified compound selected from the group consisting of compounds of the following formulas:

;

where:

-A-a - represents the group-CR102R102a-CR103R103aor CR102=CR103where CR102R102a- and-CR102= corresponds to the C2 carbon, a CR103R103aand =CR103- meet the carbon C3;

-D-D - represents the group-CHR104-CH or-CR104=C-;

-E-e - represents the group-CH2-CR110- or-CH=;

-A-E - represents the group-CR102R102a-CH2or CR102=CH, CR102R102a- and-CR102= rela is lstout carbon C2, a-CH2and=CH - meet the carbon C1;

-G-G - represents the group-CR106R106a-CHR107or CR106=CR107-where is CR106R106a- and-CR106= match the C6 carbon, a-CHR107and =CR107- meet the carbon C7;

-J-J - represents the group-CR108-CR109- or-C=C-, CR108- corresponds to the carbon C8, a-CR109- corresponds to the carbon C9;

-L-L - represents the group-CR111R111a-CH2or CR111-CH-, CR111R111a- and-CR111= match carbon C11, a-CHR2and =CH - meet the carbon C12;

-J-L - represents the group-CR109-CR111R111a- or-C=CR111-where is CR109and C= correspond to the C9 carbon, a CR111RStreet 111A- and-CR111- meet the carbon C11;

-M-M - represents the group-CR114-CH2- or-C=CH-, CR114and C= correspond to C14 carbon, a-CH2- and-C=N - correspond to the carbon C15;

J-M - represents the group-CR108-CR114- or-C=C-, CR108- corresponds to the carbon C8, a-CR114- corresponds to the carbon C14;

-Q-Q - represents the group-CR120R120a-CR119R119aor CR120=CR119-where is CR120R120a- and-CR120=CR119corresponds to C20 carbon, a CR119R119aand =CR119- meet the carbon C19;

-Q-T - gr is the PUR-CR 119R119a-CHR118or CR119=CR118-where is CR119R119a- and-CR119= match carbon C19 a-CHR118and =CR118- meet the carbon C18;

R102represents hydrogen, alkyl, alkenyl or quinil;

R102arepresents hydrogen; or R102arepresents the relationship between the carbon atom C2 and the carbon atom C3 in if-a-a-represents the group-CR102=CR103-and-a-E - represents the group-CR102R102a-CH2-or the relationship between the carbon atom C1 and a carbon atom C2 if-a-E - represents the group-CR102=CH-, a-a-a - represents the group-CR102R102a-CR103R103a-;

R103represents hydrogen, hydroxy, protected hydroxy, R130O-, R130C(O)O-, R130OC(O)O - or R103taken together with R103aforms oxo provided that-a-a - represents-CR102R102a-CR103R103a-when R103together with R103aform oxo;

R103arepresents hydrogen, or R103atogether with R103form oxo, provided that-a-a - represents-CR102R102a-CR103R103a-when R103atogether with R103form oxo;

R104represents hydrogen, alkyl, alkenyl or quinil;

R106represents hydrogen, hydroxy or protected hydroxy, or together with R106 forms an oxo, or together with R106and the carbon atom to which they are bound, form cyclopropyl, cyclobutyl or cyclopentene ring, provided that-G-G - represents the group-CR106R106a-CR107R107a-if R106together with R106aform oxo;

R106arepresents hydrogen, hydroxy or protected hydroxy, or together with R106forms an oxo, or together with R106and the carbon atom to which they are bound, form cyclopropyl, cyclobutyl or cyclopentene ring; or RAtogether with R107aand carbon atoms to which they are linked, form cyclopropyl, cyclobutyl or cyclopentene ring;

R107represents hydrogen; hydroxycarbonyl; lower alkyl, alkenyl, quinil, aryl, heteroaryl or aralkyl; halogenated; hydroxyalkyl; alkoxyalkyl; lower alkanoyl, alkanoyl, alkanoyl, ariail, heteroaryl or arkanoid; lower alkoxycarbonyl, ascenoccary, alkyloxyaryl, aryloxyalkyl, heteroarylboronic or arelaxation; lower alkanoate, ulceneili, alkinity, allolio, heteroaromatic or arachnoidea; lower alkylthio, alkanity, alkylthio, aaltio, heteroaromatic or kalkilya; carbamyl; alkoxycarbonyl; or cyano; or

R107together with R106aand the atoms of carbon is a, to which they are linked, form cyclopropyl, cyclobutyl or cyclopentene ring; or

R107and R114taken together with the carbon atoms C7, C8 and C14 form γ-lactone;

R108represents hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, quinil, R140O-, R140C(O)O-, R140OC(O)O-; or represents a bond between a carbon atom C8 and carbon atom C9 if-J-J - represents the group-C=C-, a-J-M - represents the group-CR108-CR114-; or represents a bond between a carbon atom C8 and carbon atom C14 if-J-M - represents the group-C=C-and-J-J - represents the group-CR108-CR114-;

R109represents hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, quinil, R150O-, R150C(O)O-, R150OC(O)O-; or represents a bond between a carbon atom C9 and carbon atom C11 if-J-L - represents the group-C=CR111-and-J-J - represents the group-CR108-CR109-; or represents a bond between a carbon atom C9 and carbon atom C8 if-J-J - represents the group-C=C-, a-J-L - represents the group-CR109-CR111R111a-;

R110represents hydrogen or methyl;

R111represents hydrogen, hydroxy or protected hydroxy, or R111together with R111aform oxo, provided that-J-L - represents g is the SCP-CR 109-CR111R111a-, a-L-L - represents the group-CR111R111a-CH2-if R111together with R111aforms an oxo;

R111arepresents hydrogen or together with R111forms an oxo, provided that-J-L - represents the group-CR109-CR111R111a-, a-L-L - represents the group-CR111R111a-CH2-if R111together with R111aforms an oxo; or R111arepresents the relationship between the carbon atom C11 and the carbon atom C9 if-J-L - represents the group-C=CR111a-L-L - represents the group-CR111R111a-CH2or R111arepresents the relationship between the carbon atom C11 and the carbon atom C12 if L-L-represents the group of CR111=CH, a-J-L - represents the group of CR109RA-CR111RStreet 111A-;

R114represents hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, quinil, R160O-, R160C(O)O-, R160OC(O)O-; or R114and R107together with the carbon atoms C7, C8 and C14 form γ-lactone; or R114represents the relationship between the carbon atom R14 and the carbon atom C8 if-J-M - represents the group-C=C-and-M-M - represents the group-CR114-CH2-; or R114represents the relationship between the carbon atom R14 and the carbon atom C15 if-M-M - represents the group-C=CH-, and-J-M - represents the group-CR 108-CR114-;

R118represents hydrogen, alkyl, alkenyl, quinil, aryl, heteroaryl, alkylthio, alkanity or cyano;

R118arepresents hydrogen or a bond between the carbon atom and C18 carbon atom C19 if-Q-T - represents the group-CR118=CR119-, a Q-Q - represents a group CR119R119a-CR120R120a-;

R119represents hydrogen, alkyl or alkenyl;

R119arepresents hydrogen or a bond between the carbon atom C19 and carbon atom C20 if-Q-Q - represents the group-CR120=CR119-, a-Q-T - represents a group CR119R119a-CR118R118a-; or the relationship between the carbon atom C19 and carbon atom C18 if-Q-T - represents the group-CR119=CR118-, a-Q-Q - represents a group CR119R119a-CR120R120a-;

R120represents hydrogen, hydroxy, protected hydroxy, or together with R120aforms an oxo; provided that-Q-Q - represents a group CR119R119a-CR120R120a-if R120together with R120aform oxo;

R120arepresents hydrogen or together with R120forms an oxo; provided that-Q-Q - represents a group CR119R119a-CR120R120a-if R120atogether with R120form oxo; and

R130, R140, R150and R160independent pre which constitute the alkyl, alkenyl, quinil, aryl or heteroaryl.

More preferably, this compound corresponds to the compound of formula C-3 wherein R107represents hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoate, lower alkylthio, carbamyl, or R107taken together with R106aand carbon atoms to which they are linked, form cyclopropyl ring; R106represents hydrogen, hydroxy, or taken together with R106aand the carbon atom to which they are bound, form cyclopropyl ring; R106arepresents hydrogen, hydroxy or, together with R106and the carbon atom to which they are bound, form cyclopropyl ring; or R106ataken together with R107and carbon atoms to which they are linked, form cyclopropyl ring; a R120is keto.

For discussion 13,17-condensed ring compounds described in this application uses the following definitions.

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, and substituted (in particular, the aryl), unsubstituted or heterogametes.

The term "lower al is anoil" means radical, preferably originating from direct alkyl having from 1 to 7 carbon atoms, and is associated with the original molecular part through a carbonyl group. Especially preferred are formyl and acetyl.

The term "lower alkoxycarbonyl" means radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms, and is associated with the oxygen atom with the specified oxygen atom attached to the original molecular part, by a carbonyl group. Especially preferred are methoxycarbonyl, etoxycarbonyl, isopropoxycarbonyl and n-hexyloxybenzoyl.

The term "lower alkenyl" means alkanniny radical having from 2 to 6 carbon atoms, such as ethynyl, propenyl, Isopropenyl, butenyl, Isobutanol, second-butanol and tert-butanol, pentanol and hexanol. This radical can be straight or branched and substituted, unsubstituted or heterogametes. The terms "lower alkanoyl" and "lower ascenoccary" are the same as defined for the "lower alkanoyl" and "lower alkoxycarbonyl", respectively, except that they come not from direct alkyl, and from direct alkenyl. Preferably, the oxygen associated with alkenyl radical any allenxlenalee group, separated from any unsaturated carbon zinc used is and at least one methylene group.

The term "lower quinil" means alkynylaryl radical having from 2 to 6 carbon atoms, such as ethinyl, PROPYNYL, Isopropenyl, butenyl, Isobutanol, second-butinyl and tert-butenyl, pentenyl and hexenyl. This radical can be straight or branched and substituted, unsubstituted or heterogametes. The terms "lower alkanoyl" and "lower alkyloxyalkyl" are the same as defined for the "lower alkanoyl" and "lower alkoxycarbonyl", respectively, except that they come not from direct alkyl, and from direct quinil. Preferably, the oxygen associated with alkynylaryl radical any alkyloxyaryl group, separate from any unsaturated carbon of at least one methylene group.

"Aryl" portion preferably contains, either individually or with different substituents from 5 to 15 atoms, and a represents phenyl.

The term "lower alkylthio" means radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms, and is associated with the original molecular part through a sulfur atom. Especially preferred is methylthio.

The term "lower alkanoate" means radical, preferably derived from direct alkyl having from 1 to 7 carbon atoms, and linked to the carbonyl group, with the specified AC is Bonilla group attached to the original molecular part through a sulfur atom. Especially preferred is acetylthio.

The terms "lower alkanity" and "lower ulceneili" are the same as defined for the "lower alkylthio" and "lower alkanoate", respectively, except that they come not from direct alkyl, and from direct alkenyl. Preferably, the sulfur atom of any alkanity group is separated from any unsaturated carbon of at least one methylene group.

The terms "lower alkylthio" and "lower alkinity" are the same as defined for the "lower alkylthio" and "lower alkanoate", respectively, except that they come not from direct alkyl, and from direct quinil. Preferably, the sulfur atom of any alkylthio group is separated from any unsaturated carbon of at least one methylene group.

The term "carbamyl" means the radical-NH2associated with the original molecular part through a carbonyl group. Carbonilla group may be mono-substituted or di-substituted, and the substituents can be alkyl, alkanniny, alkynylaryl and aryl radicals.

Groups defined above can be unsubstituted or optionally substituted. Such additional substituents can be alkyl, alkenyl, quinil, aryl, heteroaryl, carboxy, such as alkoxy, carboxylic, the cyl, acyloxy, halogen, such as chlorine or fluorine, halogenoalkane, nitro, amino, amido, keto. Groups defined above as well as additional substituents may also contain oxygen, sulfur, phosphorus and/or nitrogen.

In the present description, "Me" means methyl; "Et" means ethyl; "AC" means acetyl.

In an even more preferred embodiment of the present invention a compound selected from the group consisting of compounds having the following formulas:

;

;

;

;

;

;

;

;

;

;

;

; and

where R107, R106, R106aand R120defined above. Preferably, R107represents hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoate, lower alkylthio, carbamyl, or together with R106aand carbon atoms to which they are linked, form cyclopropyl ring; R106represents hydrogen, hydroxy, or together with R106a and the carbon atom to which they are bound, form cyclopropyl ring; R106arepresents hydrogen, hydroxy, or together with R106and the carbon atom to which they are bound, form cyclopropyl ring, or together with R107and carbon atoms to which they are linked, form cyclopropyl ring; a R120is keto. Even more preferably, if this connection, moreover, corresponds to the compound of formula C-5.

In an even more preferred embodiment of the invention the compound of formula C-3 are selected from the group consisting of the following compounds:

In the most preferred embodiment of the invention in this connection is the methyl-2,2,3A,4,6,7,9,10,-11,11a,12,13-dodecahydro-3αβ,11αβ-dimethyl-1,9-dioxo-1H-pentalen[1,6A-a]phenanthrene-6α-carboxylate:

This compound of formula C-1 is particularly preferred chromatographic marker when receiving epoxyoctane.

New connections discussed in the us is oasam description can be obtained in the form of their salts.

Getting new connections

Basically, the new compounds described directly above, can be obtained by reaction of the steroid with 20-spiroxamine ring and steroid nucleus described above, with trihalomethanes alanovoy acid. The preferred reagent for this reaction is a salt of an alkali metal and used alanovoy acid. Particularly preferably, the reagent for the reaction consisted of 3-florexpo acid and a salt of an alkali metal and of this acid, such as triptorelin potassium. In addition, a desiccant, such as triperoxonane 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-, -D-D-, -e-E-, -a-E-, -G-G-, -J-J-, -L-L-, -J-L-, -M-M-, -J-M-, -Q-Q-and-Q-T is defined above. Such starting compound can be obtained and/or selected by the methods similar to the methods described in Scheme 1 for the previously discussed method for the synthesis of epoxyoctane. Alternatively, the source compounds are commercially available.

The initial concentration of the steroid compounds of formula C-3 is preferably at the very the least about 0.1% by weight of the entire reaction mixture, more preferably from about 2% to about 20 wt. -%, and even more preferably from about 5% to about 15% of the mass. Preferably, trigalogenmetany acid is present in excess. If you are using triperoxonane anhydride, its number should be at least about 3% by weight of the entire reaction mixture, more preferably from about 5% to about 25 wt. -%, and most preferably from about 10% to about 15% of the mass.

In addition, the reaction temperature should not exceed room temperature (22°). Preferably, the reaction temperature is from about 40°to 100°S, more preferably from about 50°C to 80°S, even more preferably from about 60°C to 70°and most preferably from about 60°to 65°C. In the reaction with increasing the reaction temperature to about 70°or better, producing a higher amount of by-product C14-lactone. 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 of the invention, the reaction time is from about one hour to two hours, and the reaction temperature is maintained at about the olo 60° C.

Negativeone scheme S-1 illustrates a particularly preferred variant of this method is:

Scheme S-1.

Steroids with condensed rings having different substituents in different positions of the steroid can be obtained in accordance with negativeone reaction schemes. Additional procedures and methods not specifically described in this application and which can be used for the introduction of different substituents at different positions of the steroid, known to every expert. The starting compounds can be either steroid with a condensed ring, or the steroid 20-pyroxenoid ring. For ease of description in the case when the source connection is the steroid with condensed rings, in the following reaction schemes are specific steroids or group of steroids as illustrative of the parent compounds. However, it should be noted that in the same series of reactions using as the source connection of another steroid with condensed rings may be produced by other steroid derivatives with condensed rings, or their equivalents. Similarly, for ease of description in the case when the source connection is the steroid 20-pyroxenoid ring, the quality is as the source of some specific compounds are used steroids with 20-pyroxenoid ring. It should be noted that in the same series of reactions using as starting compound other steroid 20-pyroxenoid ring, can be produced other steroid derivatives with 20-pyroxenoid ring, or their equivalents.

Steroids having carbonisation Vice C7, can be obtained by the saponification reaction of the steroid with alkoxycarbonyl Vice C7, such as compound of formula s-1. The saponification reaction can be carried out by treating the source of the steroid alkaline reagent, such as potassium hydroxide or sodium 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 in the absence of water. As illustrated in Scheme S-2, the reaction of saponification of compounds of formula C-1 gives the carboxylic acid of formula-101.

Steroids having carbonwater the substituents at C7, which is not metanotum, can be obtained using carboxylic acids, such as 101, as starting compounds. The handling of such carboxylic acids alkylating agent, such as alkylhalogenide, in the presence of a base (such as sodium bicarbonate, sodium carbonate, potassium bicarbonate or triethylamine) in a solvent such as dimethylformamide, leads to the right is false of esters. Examples of suitable alkylating agents are ethyliodide, ethylbromide, isopropylated, hexalite, benzylbromide, allride etc.

For the synthesis of carbamino a suitable starting compound is also carboxylic acid S-101. Treatment of acid CHLOROFORMATES, such as isobutylparaben or ethylchloride, in the presence of base yields a mixed anhydride. Treatment of the mixed anhydride with the amine (such as dimethylamine, methylamine or Benjamin) leads to the formation of carbamyl, where R1and R2are substituents on different amines have had.

Scheme S-2.

Some modifications in position C7 made using unsaturated ketones, such as compounds of formula C-105 (shown below in Scheme S-3) as starting compounds. Sulfides synthesized by attaching suitable thiols in basic conditions. Examples of suitable thiols are methyl mercaptan, ethyl mercaptan, etc. Suitable bases are piperidine, triethylamine, etc.

Treatment of unsaturated ketones (such as compound of formula S-105) dialkanolamine acids such as teoksessa acid, leads to the formation of C7-tjalling compounds, such as acetylthio.

Condensed C6,C7-cyclopropyl Deputy can be injected PU who eat processed unsaturated ketones (such as compound of formula S-105) matilida dimethylsulfoxide, which is formed by processing halide trimethylsulfoxonium a suitable base (such as sodium hydride) in a suitable solvent.

These various schemes of synthesis is illustrated in Scheme S-3, shown below:

Scheme S-3.

Steroids containing C6-spirocyclopropane ring, synthesized by the methods described below in Scheme S-4. First anony, such as compound of formula S-110, protect in the form of a simple ester enol C3 by processing complex ortho-ester, such as triethylorthoformate or triethylorthoformate, in the presence of acid, such as p-toluensulfonate acid. Received a simple enol ether is treated with a reagent of Vilsmeier formed in situ by the addition of phosphorus oxychloride to dimethylformamide with the formation of the formyl compounds, such as compound of formula S-112. The restoration of the formyl group carried out using a hydride reducing agent such as lithium tri-tert-butoxyaniline hydride in a solvent such as tetrahydrofuran. This leads to the intermediate alcohol, which upon treatment with acid eliminates water with the formation of the 6-methylene compounds, such as compound of formula S-113. A suitable acid is hydrochloric acid in the aquatic environment. Treatment of 6-methylene connection is in the diazomethane leads to the formation of intermediate pyrazoline, which decomposes upon heating to obtain the product, such as spirocyclopropane compound of formula C-114. Secure simple enol ether (such as compound of formula S-111) is a versatile intermediate connection and processing hydride reducing agent, such as borohydride sodium, followed by acid hydrolysis leads to the production of hydroxy compounds, such as compounds of formula C-115 and C-116. These various stages of synthesis is illustrated below in Scheme S-4:

Scheme S-4.

Steroids having the hydroxy-Vice C6 and hard-essential Vice C7, can be synthesized by the methods illustrated below in Scheme S-5. Ester (such as compound of formula s-1) protect the carbonyl NW through the formation of a simple ester of 3,5-gianola (such as compound of formula S-117) using complex ortho-ester, such as triethylorthoformate or triethylorthoformate, in the presence of acid. A suitable acid is p-toluensulfonate acid. The treatment is simple ester enol oxidant such as meta-chloroperoxybenzoic acid, leads to the formation of hydroxy-compounds, such as compound of formula S-118.

Scheme S-5

In Scheme S-6 illustrates the introduction of a double bond in position S1 is the 2 steroid. This can be achieved by processing the desired steroid (such as compounds of formula C-1, C-108 and C-114) with a suitable oxidant, such as DICHLORODIFLUOROMETHANE, in a suitable solvent (such as dioxane) at temperatures reaching up to the boiling point. This method can be obtained unsaturated compounds C1-C2, such as compounds of formulas C-127, 128 and 129.

Scheme S-6.

In Scheme S-7 illustrates the introduction of double bonds in the condensed ring. Steroid (such as compound of formula S-114) handle complex orthoevra (such as triethylorthoformate or triethylorthoformate) in the presence of an acid catalyst such as p-toluensulfonate acid) to obtain the simple enol ether, where the C3 carbonyl is protected. In the case of the compounds of formula C-114, because the position C6 is completely replaced, educated simple enol ether is an ether of enol C2-C3 (such as compound of formula S-131). Treatment of the enol ether of a strong base, such as diisopropylamide lithium at low temperature (-78°C to -30°C), with subsequent processing selenium agent such as phenylsalicylate, leads to the formation of the selenium derivative, such as Obedinenie formula-132. Oxidation of selenium derived oxidant, 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, leads to the elimination of selenium group and the introduction of a double bond. The simple hydrolysis of the enol ether yields a ketone, such as compound of formula S-134.

Scheme S-7.

In Scheme S-8 illustrates the synthesis of isomers with the double bond of the compounds of formula C-1. Processing of various pyroxenoid compounds shown in Scheme S-8, potassium acetate, triperoxonane anhydride and triperoxonane acid under conditions similar to the conditions of synthesis of the compounds of formula C-1, leads to the formation of compounds of formula C-121 and C-123.

Scheme S-8.

In Scheme S-9 illustrates an alternative method of synthesis of isomers with double bonds for this family of steroids. In the prior northward (such as compound of formula s-24, the synthesis of which is described above), already containing condensed ring, introducing the condensed C6-C7-cyclopropane using chemical methods described above in Scheme S-3 for the synthesis of compounds such as compound of formula S-108 and S-109.

Scheme S-9.

where X represents halogen.

Condensed ring steroids having an aromatic ring And may be obtained by treatment with steroids, such as steroids, described in P.Compain, et al., Tetrahedron, 52(31), 10405-10416 (1996) (which is introduced in the present description by reference), triperoxonane acid, potassium acetate and triperoxonane anhydride, mainly, in a manner analogous to the previously discussed for Scheme S-1.

It is expected that these new condensed ring steroids containing an aromatic ring and 3-hydroxy-Deputy, exposed to all the chemical reactions that are typical for phenols. In Scheme S-10 illustrates the synthesis of a simple ester 3-phenol of this steroid with condensed rings. In particular, it is assumed that the processing of these phenolic compounds base and alkylhalogenide or alkylsulfonates leads to the production of the corresponding complex ester of phenol. Cm. work Feuer & Hooz, In The Chemisty of the Ether Linkage, Patai (Ed.), Interscience: New York, pp.446-450, 460-468 (1967); and Olson, W.T., SOC., 69, 2451 (1947), which are introduced in the present description by reference.

Scheme S-10.

Similarly, the Scheme S-11 illustrates the synthesis of ester 3-phenol of the steroid with condensed rings having aromatic ring and 3-hydroxy-Zam the Titel. In particular, it is assumed that the processing of these phenolic compounds with carboxylic acid anhydride or carboxylic acid halide leads to the production of the corresponding complex ester of phenol. Cm. work March, J., Advanced Organic Chemistry, Wiley: New York, pp.346-347 (1985), which is introduced in the present description by reference.

Scheme S-11.

Scheme S-12 illustrates the synthesis of carbonate 3-phenol of the steroid with condensed rings having aromatic ring and 3-hydroxy-Deputy. In particular, it is assumed that the processing of these phenolic compounds by alkylhalogenide leads to the production of the corresponding carbonate phenol. Cm. work March, J., Advanced Organic Chemistry, Wiley: New York, pp.346-347 (1985), which is introduced in the present description by reference.

Scheme S-12.

Scheme S-13 illustrates the synthesis of ortho-allyl-substituted phenyl derivatives of the steroid with condensed rings having aromatic ring and 3-hydroxy-Deputy. In particular it is expected that the processing of these phenolic compounds base and allergologicum leads to the production of the corresponding allylanisole ether. This allergenicity ether have to give a mixture of ortho-allyl-substituted phenyl derivatives after thermal rearrangement. Cm. mon the graphy Shine, H., J.Aromantic Rearrangements; Reaction Mechanisms in Organic Chemistry, Monograph 6, American Elsevier; New York, pp.89-120 (1967), which is introduced in the present description by reference.

Scheme S-13.

Scheme S-14 illustrates the synthesis of ortho-dialkylamines substituted phenyl derivatives of the steroid with condensed rings having aromatic ring and 3-hydroxy-Deputy. In particular, it is expected that the treatment of these compounds with alcohol and acid leads to the production of the corresponding ortho-dialkylamines phenyl derivatives. See, for example, the work Calcott, W.S., SOC., 61, 1010 (1939), which is introduced in the present description by reference.

Scheme S-14.

Scheme S-15 illustrates the allylic oxidation of the steroid with condensed rings having aromatic ring and 3-hydroxy-Deputy. In particular, these compounds can be oxidized in the allyl position by reaction with selenium dioxide and tert-butylhydroperoxide with the formation of the corresponding alcohol. As a result of dehydration of the alcohol is prepared the corresponding olefin. See, for example, the work Schmuff, N.R., J.Org.Chem., 48, 1404 (1983), which is introduced in the present description by reference.

Scheme S-15.

Scheme S-16 illustrates for the ITU 3-carbonyl groups and the restoration of the 20-carbonyl group of new steroids with condensed rings of the present invention. In particular, these compounds can be subjected to reaction with trialkylaluminium and acid with the formation of a simple ester 3-enol. This simple ester of 3-enol can then be subjected to reaction with sodium borohydride recovery-20 carbonyl group to the corresponding C-20 alcohol. Treatment With-20-alcohol acid and water provides release ether 3-enol with the formation of 3-keto-derivative.

Scheme S-16.

Scheme S-17 illustrates the hydrogenation of olefinic bonds in new steroids with condensed rings of the present invention. In particular, it is assumed that the hydrogenation proceeds Paladino. First, saturated C-6,C-7 double bond, and then saturated C-8,C-14-double bond.

Scheme S-17.

Scheme S-18 illustrates the rearrangement protected 11α-hydroxy-steroid with condensed rings of the present invention. In particular, first, 11α-hydroxy group protects a suitable protecting group, such as 2-Metacritic-semesterby ether (MEM-ether). It is expected that the treatment protected 11α-hydroxy-steroid salt of an alkali metal and trigalogenmetany acid in the presence of acid anhydride leads to pregreplace lactone part in these molecules, as illustrated below. Remove MEM-JFK and the RA 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 alkylation of 19-regulations of new steroids with condensed rings of the present invention. In particular, this first connection is turned into a simple ester of 3-alkylene, as shown in figure S-16. The treatment is simple ester 3-alkylene diisopropylamide lithium (LDA) followed by treatment with alkylhalogenide leads to the formation of 19-Olkiluoto derived. As a result of hydrolysis of the protective group of a simple ester of 3-alkylene get 3-keto-derivative.

Scheme S-19.

Scheme S-20 illustrates a simple transformation of the methyl ether of estrone in the appropriate spirolactone. Treatment of the lactone trihalomethanes alanovoy acid, preferably in the presence of salts of alkaline metal used alanovoy acid, in the reaction conditions described above leads to the rearrangement of the lactone to the corresponding steroid with condensed rings. See, for example, Otsubo, K., Tetrahedron Letters, 27(47), 5763 (1986).

Scheme S-20.

The new compounds described in this application can be also subjected to the methods of biological transformation, similar to the methods described previously, with other new steroid is in the condensed rings, such as steroids, with 9α-, 9β-, 11αor 11β-hydroxy-Deputy, as well as other gidroksilirovanii steroids with condensed rings. If necessary, such gidroksilirovanii steroids can then be oxidized by elimination hydroxy-Deputy for the introduction of the olefinic double bond, such as Δ9,11-olefinic double bond.

Based on the above, we can say that we have achieved some of the objectives of the present invention and obtained other primary results.

It should be noted that although in the above described compositions and methods can be made various changes, not beyond the scope of the present invention, however, all the 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. Obtaining methyl-2,3,3A,4,6,7,9,10,11,11a,12,13-dodecahydro-3Aβ,11aβ-dimethyl-1,9-dioxo-1H-pentalen[1,6A-a]phenanthrene-6α-carboxylate (Compound C-1)

In the clean and dry reactor equipped with a mechanical stirrer, a refrigerator, a thermocouple and a casing for heating, was added potassium acetate (6.7 g, 7.1 mmol; Sigma-Aldrich 5128LG) Then the reactor was sequentially added triperoxonane acid (25,0 ml, 8.1 mol; Sigma-Aldrich 7125MG) and triperoxonane anhydride (4.5 ml, was 31.0 mmol; Sigma-Aldrich 11828PN). Then the solution was kept for 30 minutes at a temperature of from 25 to 30°C.

Prior reagent TFA/TFA anhydride was added to 5.0 g (9.6 mmol) of 7-Metelitza-17-hydroxy-11α-(methylsulphonyl)oxy-3-oxo-17α-pregna-4-ene-7α,21-in primary forms, γ-lactone:

which was obtained by the method described in Example 36. The resulting mixture was heated for 60 minutes at 60°and the degree of transformation is periodically evaluated by TLC and/or HPLC. At the completion of the reaction (approximately 60 minutes), the mixture was transferred into a 1-necked flask and concentrated under reduced pressure at 50°until then, until a thick suspension.

The resulting suspension was diluted with 150 ml of ethyl acetate and 80 ml of a mixture of water/saturated salt solution. The mixture is then left for phase separation and the aqueous layer was re-extracted with 80 ml of ethyl acetate. The fortress of saturated salt solution was 12% of the mass. United an ethyl acetate solution once washed 12% of the mass. saturated brine (80 ml)and then 1N NaOH solution (80 ml) and, finally, 2% of the mass. saturated brine (80 ml). The mixture was left for phase separation and separated an ethyl acetate layer was concentrated to dryness with onigen the m pressure at 45° With using the device for pumping water, resulting received about 3.8 g of crude solid product. HPLC analysis of this crude product showed that the product contained about 40% (by area) of compound C-1.

Then this solid product was subjected to chromatographic purification. After chromatographic purification was obtained 210 mg of methyl-2,3,3A,4,6,7,9,10,11,11a,12,13-dodecahydro-3Aβ,11aβ-dimethyl-1,9-dioxo-1H-pentalen[1,6A-a]phenanthrene-6α-carboxylate (Compound C-1).

The data of mass spectrometry showed that the molecular weight is 380, and using data obtained with high resolution, was established formula C14H28O4. The mass spectrum obtained by the method of EI, had peak M+ at m/z 380. The mass spectrum obtained by the method of APHI, had peaks at m/z 381 (MH)+ and m/z 398 (MNH4)+. Carbon and hydrogen analyses were consistent with the proposed molecular formula.

The IR spectrum has two peaks in the region of absorption of the carbonyl: 1722 cm-1and 1667 cm-1. Peak 1722 cm-1attributed to two CARBONYLS, as13C-NMR spectrum had signals at the δ 217,7, obuslovennom saturated ketone, and when δ 172,7 due carbomethoxyamino. The absence of peak 1773 cm-1the IR spectrum indicates the loss of the lactone ring part.

Data13With ART and HETCOR NMR indicate the presence of coal the delivery of the following types: 3 carbonyl (δ 217,7, 198,4, 172,2); 4 fully-protected olefinic carbon (δ 166,3, 141,8, 139,3, 121,8); 2 marinaleminova carbon (δ 124,9, 122,0); 3 Quaternary aliphatic carbon (δ 61,1, 50,7, 39,7); 1 aliphatic methine carbon (δ 43,3); 8 methylene carbons (δ 46,0, 37,5, 34,1, 33,3, 32,9, 31,9, 23,7, 22,2) and 3 methyl carbon (δ 51,9, 23,6, 23,1).

Example X-1B. Obtaining (7α,13R,17β)-3',4',5',17-tetrahydro-14-hydroxy-17-methyl-3,5'-dioxo-γ-lactone, cyclopent[13,17]-18-norandro-4,9(11)-diene-7-carboxylic acid (Compound C-201)

In a 250 ml round bottom reactor equipped with a mechanical stirrer, a refrigerator and a casing for heating, was added potassium acetate (8,9 g, 90 mmol), triperoxonane acid (150 ml, 1,480 g/ml) and triperoxonane anhydride (33 ml, 1,487 g/ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25°With 30°C.

Prior reagent TFA/TFA anhydride, was added to 15 g (30.0 mmol) of 7-Metelitza-17-hydroxy-11α-(methylsulphonyl)oxy-3-oxo-17α-pregna-4-ene-7α,21-in primary forms, 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°S-70°C. the mixture was concentrated under reduced pressure at 50°until then, until obrazovym who stayed thick suspension. The resulting suspension was diluted with 100 ml ethyl acetate and 2 times washed about 20% water/saturated salt solution each time (80 ml), 1 1N solution of sodium hydroxide (80 ml), and then 1 time a mixture of about 20% water/saturated salt solution (80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated, resulting in received about 18 g of crude material.

This product twice was purified by column chromatography and was about 3 g of pure (7α,13R,17β)-3',4',5',17-tetrahydro-14-hydroxy-17-methyl-3,5'-dioxo-γ-lactone, cyclopent[13,17]-18-norandro-4,9(11)-diene-7-carboxylic acid (Compound C-201).

Example X-1C. Getting [13S,17β]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopentane[13,17]gona-1,3,5(10)-triene-5'-[2 N]-she (Connection-202)

In a 250 ml round bottom reactor equipped with a mechanical stirrer, a refrigerator and a casing for heating, was added potassium acetate (6 g, with 61.1 mmol), triperoxonane acid (150 ml, 1,480 g/ml) and triperoxonane anhydride (26 ml, 1,487 g/ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25°With 30°C.

Prior 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 Aldona; G.D.Searle & Co.):

The resulting mixture was heated for about 1-1 .5 hours at 60°S-70°C. the mixture was concentrated under reduced pressure at 50°until then, until it formed a thick slurry. The resulting suspension was dissolved in 100 ml ethyl acetate and 2 times washed about 20% water/saturated salt solution each time (80 ml), 1 1N solution of sodium hydroxide (80 ml), and then 1 time a mixture of about 20% water/saturated salt solution (80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated to dryness under reduced pressure at 50°With, which got about 20 g of the crude wet product.

This product twice was purified by column chromatography and was about 125 g of pure [13S,17β]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopentane[13,17]gona-1,3,5(10)-triene-5'-[2 N]-she (Connection-202).

Example X-1D. Getting [13S,17β]-3',4'-dihydro-3-hydroxy-9,17-dimethylcyclopentane[13,17]gona-1,3,5(10),6-tetraen-5'-[2 N]-she (Connection-203)

In a 250 ml round bottom reactor equipped with a mechanical stirrer, a refrigerator and a casing for heating, was added potassium acetate (6 g, with 61.1 mmol), triperoxonane acid (150 ml, 1,480 g/ml) and triperoxonane anhydride (26 ml, 1,487 g/ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25°d is about 30° C.

Prior 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 (also known as Δ-9,11-Aldona):

which was obtained from 3-methoxy-3,5,9(11)-androstatriene-17-she (decision Upjohn). The resulting mixture was heated at about 60°S-70°C for about 1-1,5 hours. This mixture was concentrated under reduced pressure at 50°until then, until it formed a thick slurry. The resulting suspension was diluted with 100 ml ethyl acetate and 2 times washed with a mixture of about 20% water/saturated salt solution each time (80 ml), 1 1N solution of sodium hydroxide (80 ml), and then 1 time about 20% water/saturated salt solution (80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated to dryness under reduced pressure at 50°With, which got about 18 g of the crude wet product.

This product twice was purified by column chromatography and was about 340 g of pure [13S,17β]-3',4'- dihydro-3-hydroxy-9,17-dimethylcyclopentane[13,17]gona-1,3,5(10),6-tetraen-5'[2 N]-she (Connection-203).

Example X-1E. Getting [13S,17β]-3',4'-dihydro-17-Methylcyclopentane-[13,17]-18-norandro-4,6,8(14)-triene-3,5'[2 N]-dione (Compound C-204)

In round-bottom 250 m of the reactor, equipped with a mechanical stirrer, a refrigerator and a casing for heating, was added potassium acetate (8 g, of 81.5 mmol), triperoxonane acid (150 ml, 1,480 g/ml) and triperoxonane anhydride (33 ml, 1,487 g/ml). The resulting solution was stirred for about 10 minutes at a temperature of from about 25°With up to about 30°C.

Prior reagent TFA/TFA anhydride, was added to 15 g (to 44.0 mmol) of 17-hydroxy-3-oxo-17α-pregn-4,6-diene-21-carboxylic acid, γ-lactone (also known as canrenone; G.D.Searle & Co.):

The resulting mixture was heated for about 1-1,5 hours at about 60°S-70°C. the mixture was concentrated under reduced pressure at 50°until then, until it formed a thick slurry. The resulting suspension was dissolved in 100 ml ethyl acetate and 2 times washed with a mixture of about 20% water/saturated salt solution each time (80 ml), 1 1N solution of sodium hydroxide (80 ml), and then 1 time a mixture of about 20% water/saturated salt solution (80 ml). The crude product was dried with magnesium sulfate, filtered and concentrated to dryness under reduced pressure at 50°With, which got about 18 g of the crude wet product.

This product twice was purified by column chromatography and received about 2.2 g of pure [13S,17β]-3',4'-dihydro-17-Methylcyclopentane[13,17]-18-norandro-4,6,8(14)-triene-3,5'[2 N]-dione (Compound C-204).

Example X-2. Receive:

To a stirred cold (0° (C) a solution of 11α-hydroxykynurenine (3.6 g, 10 mmol) and triethylamine (1.2 g, 12 mmol) in methylene chloride (20 ml) was added methanesulfonamide (1.1 g, 10 mmol). The mixture was stirred with cooling for 3 hours and left to warm to room temperature. Stirring was continued up until thin layer chromatography indicated the completion of reaction. Then the mixture was diluted with ethyl acetate and was extracted with water, aqueous 5% sodium bicarbonate solution and water and then dried with sodium sulfate. The desiccant was filtered and the filtrate was concentrated in vacuo to obtain the crude nelfinavir C-136, which can be used in the following stages:

The solution nelfinavir C-136 (4.3 g, 10 mmol) was subjected to reaction with triperoxonane acid (25 ml), triperoxonane anhydride (4.5 ml) and potassium acetate (6.7 g, 7.1 mmol) by the method described for the synthesis of Compound C-1 in Example X-1. The crude product was isolated in a manner analogous to that described for Compound C-1 in Example X-1 was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents. Then the obtained product was purified by recrystallization from alcohol, Speer is a and water or ethyl acetate and hexane to obtain tetraena C-105.

Example X-3. The connection is:

Mesilate C-138:

synthesized and identified (by the method described in Example X-2 for the synthesis of nelfinavir With-136) using 11α,17-dihydroxy-3-oxo-17-oxo-pregn-4-ene-21-carboxylic acid, γ-lactone (3.6 g, 10 mmol), triethylamine (1.2 g, 12 mol) and methanesulfonamide (1.1 g, 10 mmol) in methylene chloride (20 ml). Selected so mesilate C-138 can be used in the next stage.

The solution nelfinavir C-138 (4.4 g, 10 mmol) was subjected to reaction with triperoxonane acid (25 ml), triperoxonane anhydride (4.5 ml) and potassium acetate (6.7 g, 7.1 mmol) by the method described for the synthesis of Compound C-1 in Example X-1. The crude product With a-110 was isolated in a manner analogous to that described for Compound C-1 in Example X-1 was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents. Then the obtained product was purified by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane.

Example X-4. Obtain 3',4',5',17-tetrahydro-17β-methyl-3,5'-doxiciclina[13R,17]-18-norandro-4,8,14-triene-7α-carboxylic acid (Compound C-101)

A solution of Compound s-1 (3.8 g, 10 mmol) and 1N aqueous solution of hydroxy is and sodium (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 under vacuum and the residual aqueous layer three times were extracted with ethyl acetate. Then the aqueous layer was acidified using 1N solution of hydrochloric acid and three times were extracted 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 carboxylic acid-101 was led by treatment with ethyl acetate and recrystallized from ethyl acetate and hexane or methanol or ethanol and water.

Example X-5. Getting 1-methylethyl-3',4',5',17-tetrahydro-17β-methyl-3,5'-doxiciclina[13R,17]-18-norandro-4,8,14-triene-7α-carboxylate (Compound C-102)

A mixture of sodium bicarbonate (3.5 g) and a solution of carboxylic acid C-101 (3.7 g, 10 mmol) and Isopropylamine (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 vacuum. The resulting crude isopropyl ester S-102 was led by treatment with ethyl acetate and the and alcohol and was purified by chromatography on silica gel, and then recrystallized from ethyl acetate and hexane or alcohol, or alcohol and water.

Example X-6. Getting ethyl-3',4',5',17-tetrahydro-17β-methyl-3,5'-doxiciclina[13R,17]-18-norandro-4,8,14-triene-7α-carboxylate

A mixture of sodium bicarbonate (3.5 g) and a solution of carboxylic acid C-101 (3.7 g, 10 mmol) and ethyliodide (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 vacuum. The residual crude ethyl ester of s-103 was led by treatment with ethyl acetate or alcohol and was purified by chromatography on silica gel and then recrystallized from ethyl acetate and hexane or alcohol, or alcohol and water.

Example X-7. Getting hexyl-3',4',5',17-tetrahydro-17β-methyl-3,5'-doxiciclina[13R,17]-18-norandro-4,8,14-triene-7α-carboxylate (Compound C-104)

A mixture of sodium bicarbonate (3.5 g) and a solution of carboxylic acid C-101 (3.7 g, 10 mmol) and n-hexylidene (3 ml) in dimethylformamide (35 ml) was stirred overnight at room temperature. The reaction mixture was poured into water and adny the 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 vacuum. The residual crude n-hexyl ester C-104 was led by treatment with ethyl acetate or alcohol and was purified by chromatography on silica gel and then recrystallized from ethyl acetate and hexane, either from the alcohol or alcohol and water.

Example X-8. Obtain 3',4'-dihydro-17-methyl-7α(methylthio)-cyclopent[13,17]-18-norandro-4,8,14-triene-3,5'(2 N)-dione (Compound C-106)

The solution tetraena C-105 (3.2 g, 10 mmol) in methanol (40 ml) and piperidine (4 ml) was cooled to 5°C. Then missed a gaseous mercaptan until then, until there was an increase in the mass 7, the pressure Vessel was tightly closed 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 the air. Methylthiophenyl C-106 was purified by recrystallization from methanol or ethyl acetate and hexane. See, for example, the procedure described in the work A.Karim & ..Brown, Steroids, 20, 41 (1972), which is introduced in the present description by reference.

Example X-9. Getting 7α (acetylthio)-3,4'-dihydro-17-Methylcyclopentane[13,17]-18-norandro-4,8,14-triene-3,5'(2 N)-dione (Compound C-107)

The solution tetraena C-105 (3.2 g, 10 mmol) in teoksessa acid (10 ml) was heated for 1 hour at 85-95°C. Then the excess teoksessa acid was removed in vacuo and the resulting crude 7α-thioacetate C-107 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 U.S. patent 3013012, J.A.Cella & R.C.Tweit, Dec, 1961, which is introduced into the present description by reference.

Example X-10. Getting 1,2,4bR(4bR*),5,5aS*,7,7aR*,8,9,11,-12bS*-dodecahydro-7a,12b-dimethyl-10aR*-cyclopropa[1]pentalene[1,6A-a]phenanthrene-3,10-dione

and

1,2,4bS(4bR*),5,5S*,8,9,11,12,12bR*-dodecahydro-7a,12b-dimethyl-10S*-cyclopropa[1]pentalene[1,6A-a]phenanthrene-3,10-dione (Compound C-109):

To a solution of iodide trimethylsulfoxonium (1 g, 4.6 mmol) in dry dimethylsulfoxide (20 ml) was added sodium hydride (220 mg, 50%dispersion in mineral oil, 4.6 mmol). The mixture was stirred at room temperature under nitrogen atmosphere until then, until he stopped the release of hydrogen. Then was added a solution of tetraena C-105 (1.12 g, 3.5 mmol) in dimethyl sulfoxide (4 ml) and stirring continued for 4 hours under nitrogen atmosphere. The reaction mixture was diluted with water and the precipitate was filtered and air-dried. Received the product was a mixture of 6β ,7β-(Compound C-108) and 6α,7α-(Compound C-109) isomers. These isomers were separated by chromatography on silica gel, and then separate the isomers was purified by recrystallization from solvents such as ethyl acetate and hexane, alcohol or alcohol and water.

Example X-11. Obtaining the compounds of formula:

To a suspension of northward-110 (3.2 g, 10 mmol) in utilitiarian (10 ml) and anhydrous ethanol (10 ml) was added monohydrate p-toluensulfonate acid (0.05 g). The reaction mixture was stirred for 30 minutes at room temperature and was suppressed by adding a few drops of pyridine. After stirring for 5 minutes at 0°the precipitate was filtered, washed with a small amount of methanol and recrystallized from ethanol or ethyl acetate and hexane containing a trace amount of pyridine, resulting in a net simple enol ether C-111. Alternatively, after adding pyridine this reaction mixture can be treated by removing all solvent in vacuo and crystallization of the residue by adding solvents such as simple ether, ethyl acetate or hexane. Raw C-111 recrystallized as described above. See, for example, the procedure described in the work R.M.Weier & L.M.Hofmann, J.Med. Chem., 20, 1304-1303 (1977), which is introduced in this clause is a means of links.

Example X-12. The connection is:

Was carried out by the procedure described in the work R.M.Weier & L.M.Hofmann, J. Med. Chem., 20, 1304-1308 (1977), which is introduced in the present description by reference.

The reagent of Vilsmeier was obtained by adding phosphorus oxychloride (4.59 g, 30 mmol) dimethylformamide (30 ml) at 0°C. After 5 minutes was added to the solution of simple enol ether C-111 (3.5 g, 10 mmol) in dimethylformamide (5 ml) and the reaction mixture was stirred for 2 hours at 0°and over night 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 the obtained crude aldehyde-112. Purification was carried out by recrystallization from solvents such as alcohol, alcohol and water or ethyl acetate and hexane. Alternatively, the crude aldehyde C-112 was isolated from the aqueous sodium acetate solution by extraction with a solvent, such as ethyl acetate. After drying with sodium sulfate and removal of solvent the residue was purified by recrystallization or by chromatography on silica gel followed by recrystallization.

Example X-13. The connection is:

To a stirred cooled solution of tri-tert-butoxy of lithium aluminum hydride (3,05 g, 12 mmol who) in tetrahydrofuran was added aldehyde C-112 (of 3.78 g, 10 mmol). The reaction mixture was stirred at room temperature for 5 hours and was suppressed by addition of water and acetic acid, it was sure that the mixture remained slightly basic. The mixture was concentrated in vacuo and the resulting solid is suspended in ethyl acetate. The solid was filtered and the filtrate was concentrated in vacuum. The residue was dissolved in a minimal volume of a solution of acetone and water (3:1) was added aqueous acetone for acidification (pH of 1.5-2.0). Acid, the reaction mixture was stirred for 1 hour at room temperature and concentrated in vacuum. The resulting crude diene C-113 was purified by recrystallization from solvents such as alcohol, alcohol and water or ethyl acetate and hexane. Alternatively, the crude diene C-113 was purified by chromatography on silica gel and then recrystallized. See, for example, the procedure described in the work P.M.Weier & L.M.Hofmann, J.Med.Chem., 20, 1304-1308 (1977), which is introduced in the present description by reference.

Example X-14. Obtain 2',3',3 aα,4',6',10',11',11 Iα,12',13'-decahydro-3'aR,3 a,11 a-dimethyl-13'aR*-Spiro-[cyclopropane-1,7'(9 M)-[1H]pentalene[1,6A-a]phenanthrene-1'9'-dione:

To a solution of dienone S-113 (1,17 g, 0.05 mmol) in tetrahydrofuran (30 ml) was added a solution of diazomethane (0.2 g, 0.07 mmol) in a simple ether (7 ml). Receiving the hydrated reaction solution was kept at room temperature for several days. Then add acetic acid to decompose the residual diazomethane and the reaction mixture was concentrated in vacuum. The residue, which was an intermediate pyrazoline, was led from the solvent, such as acetone, hexane, ethyl acetate, or ethanol, and then recrystallized. This compound was converted into spirocyclopropane C-114. The thus obtained solid substance With a-114 was heated at 190°in vacuum and the resulting solid is recrystallized from alcohol, alcohol and water, acetone and water or ethyl acetate and hexane. Alternatively, the solution pyrazoline in acetone was treated with efratom of boron TRIFLUORIDE for about 1 hour at room temperature. Then added water and the resulting precipitate was filtered and air-dried. Purification was performed by recrystallization. See, for example, the procedure described in U.S. patent 3499891, issued March 10, F..Colton & R.T.Nicholson, which is introduced into the present description by reference.

Example X-15. The connection is:

and

The simple solution of enol ether C-111 (3.2 g, 10 mmol) in methanol (20 ml) was added borohydride sodium (38 mg). The reaction mixture was stirred for 3 hours at room temperature and was treated with 1N hydrochloric acid for 30 minutes. Then the reactions is nnow 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 Connection-116)was chromatographically on silica gel to separate them. Each of the purified alcohols C-115 and C-116 was led from solvents such as alcohol, alcohol and water, the ethyl acetate and hexane, and acetone and hexane. Alternatively, dilute the reaction mixture was extracted with ethyl acetate, the combined organic layers were dried with sodium sulfate and underlined the crude product was chromatographically as described above with obtaining the individual alcohols C-115 and C-116.

Example X-16. The connection is:

Compound s-1 (3.8 g, 10 mmol) was transformed into a simple enol ether C-117 using utilitiarian (3.2 g, 10 mmol), ethanol (10 ml) and monohydrate p-toluensulfonate acid (0.05 g) in accordance with the procedure described for the synthesis of Compound C-111 in the Example X-11.

Example X-17. Obtain methyl-3',4',5',17-tetrahydro-6α-hydroxy-17β-methyl-3,5'-dioxocyclohexa-[13R,17]-18-norandro-4,8,14-triene-7α-carboxylate (Compound C-118):

A solution of 57% m-chloroperoxybenzoic acid (of 3.64 g) in 10% aqueous dioxane (20 ml) half-neutralized 1N solution of sodium hydroxide. This solution was cooled to 0°and portions were added with stirring cold (° (C) the simple solution of enol ether C-117 (4.1 g, 10 mmol) in 10% aqueous dioxane (20 ml). The reaction mixture was stirred over night at room temperature, poured into ice water and was extracted with methylene chloride or ethyl acetate. The combined organic layers were dried with sodium sulfate. After evaporation of the solvent was obtained crude complex hydroxy-ether-118, which was purified by chromatography on silica gel. See, for example, the procedure described in the work R.M.Weier & L.M.Hofmann, J.Med.Chem., 20, 1304-1308 (1977), which is introduced in the present description by reference.

Example X-18. The connection is:

Dimethylbutyl lithium was obtained by addition of methyl-lithium (19 ml of a 1.6 m solution in simple ether, 30 mmol) to a stirred suspension of copper iodide (2.86 g, 15 mmol) in a simple ether (30 ml) at 0°in an inert atmosphere of argon. After stirring for 15 minutes in the cold dropwise within 25 minutes was added a solution of tetraena C-105 (1.6 g, 15 mmol) in tetrahydrofuran. 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 UDA the Yali in vacuum and the residue was dissolved in ethyl acetate or methylene chloride and was treated with p-toluensulfonate acid (100 mg) on 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 vacuum to obtain crude Aenon C-119. Purification was carried out by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane. Alternatively, the crude Aenon C-119 was chromatographically on silica gel, after which the product was recrystallized. See, for example, the procedure described in the work J.K.Grunwell et al., Steroids, 27, 759-771 (1976), which is introduced in the present description by reference.

Example X-19. Receive:

Ester C-120 (4,00 g, 10 mmol):

(received in accordance with the procedure described in R.M.Weier & L.M.Hofmann, J.Med.Chem., 18, 817 (1975), which is introduced in the present description by reference) were subjected to reaction with triperoxonane acid (25 ml), anhydride triperoxonane acid (4.5 ml) and potassium acetate (6.7 g, 7.1 mmol) according to the procedure described for the synthesis of Compound C-1 in Example X-1. The crude product C-121 was isolated according to the procedure described for the synthesis of Compound C-1 in Example X-1 was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents and thus obtained product was further purified by recrystallization from alcohol, the alcohol and the odes or ethyl acetate and hexane.

Example X-20. Receive:

Northward-122 (3,68 g, 10 mmol):

(received in accordance with the procedure described in U.S. patent 3499891 issued 10.03.1970, F.B.Colton & R.T.Nicholson, which is introduced into the present description by reference) were subjected to reaction with potassium acetate (6.7 g, 7.1 mmol), triperoxonane acid (25 ml) and the anhydride triperoxonane acid (4.5 ml) according to the procedure described for the synthesis of Compound C-1 in Example X-1. The crude product was isolated in accordance with the above procedure and was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents, and thus obtained product was further purified by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane.

Example X-21. Receive:

and

Connection s-125 and s-126 was synthesized according to the procedure used above for the synthesis of Compounds S-108 and s-1. To a solution of iodide trimethylsulfoxonium (1 g, 4.6 mmol) in dry dimethylsulfoxide (20 ml) was added sodium hydride (220 mg, 50% dispersion in mineral oil, 4.6 mmol). The mixture was stirred at room temperature under nitrogen atmosphere until then, until Pres who was resales hydrogen gas.

Then solution was added Tritone C-124 (1,14 g, 3.5 mmol):

in dimethyl sulfoxide (4 ml) and stirring continued for 4 hours under nitrogen atmosphere. Trianon C-124 received in accordance with the procedure described in Example X-2 to get tetraena C-105, using as starting compound canrenone instead of 11α-hydroxy-canrenone. The reaction mixture was diluted with water and the precipitate was filtered and air-dried. The product was a mixture of 6β,7β-(Compound C-125) and 6α,7α-(Compound C-126) isomers. The separation of these isomers was performed using chromatography on silica gel and separate the isomers was further purified by recrystallization from solvents such as ethyl acetate and hexane, alcohol or alcohol and water.

Example X-22. Obtain methyl-3',4',5',17-tetrahydro-17β-methyl-3,5'-doxiciclina[13R,17]-18-norandro-1,4,8,14-tetraen-7α-carboxylate (Compound C-127)

Denon C-127 was synthesized according to the procedure described in the work R.M.Weier & L.M.Hofmann, J.Med.Chem., 18, 817 (1975), which is introduced in the present description by reference. A solution of Compound s-1 (3.8 g, 10 mmol) and dichlorodicyanoquinone (2,72 g, 12 mmol) in dioxane (80 ml) was heated with stirring under reflux for 24 hours is. The reaction mixture was concentrated in vacuum, the residue is hydrolyzed with methylene chloride, filtered and the filtrate was washed with 2% sodium sulfite, 5% sodium hydroxide and a saturated solution of sodium chloride, and then dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuum. The crude product Denon C-127 was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene as eluents and thus obtained product 27 was further purified by recrystallization from alcohol.

Example X-23. Obtain 2',3',3 aα,4',6',11 Iα,12',13'-octahydro-3'aR,3 a,11 a-dimethyl-13'aR*-Spiro-[cyclopropane-1,7'(9 M)-[1H]pentalene-[1,6A-a]phenanthrene-1'9'-dione (Compound 128)

Using a procedure and process described above in Example X-22 for the synthesis of dienone C-127, northward-114 (3,48 g, 10 mmol) was converted into dienen 128 using DICHLORODIFLUOROMETHANE (2,72 g, 12 mmol) in dioxane (80 ml).

Example X-24. Getting 4bR(4bR*),5,5aS*,7,7aR*,8,9,11,-12,12bS*-decahydro-7A,12b-dimethyl-10R*cyclopropa(1)pentalene[1,6A-a]phenanthrene-3,10-dione (Compound C-129)

Using a procedure and process described above in Example X-22 for the synthesis of dienone C-127, northward-108 (3,34 g, 10 mmol) was converted into Denon C-129 using dichlorodicyanoquinone is on (2,72 g, 12 mmol) in dioxane (80 ml).

Example X-25. Receive:

To a cold (0° (C) stirred solution of carboxylic acid C-101 (3,66 g, 10 mmol) and N-methylmorpholine (1.01 g, 10 mmol) in tetrahydrofuran (35 ml) was added isobutylbarbituric (1,36 g, 10 mmol). The reaction mixture was stirred for 20 minutes at 0°C, filtered and the filtrate was concentrated in vacuum. The rest was a mixed anhydride and was suitable for use in the next stage.

Gaseous dimethylamine was barbotirovany in cold (0° (C) the solution of the mixed anhydride (4.6 g, 10 mmol) in tetrahydrofuran (40 ml) in a pressure vessel. After 15 minutes, the pressure vessel was tightly closed 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 was cooled to 0°C. After warming to room temperature the pressure in the vessel was reduced to atmospheric and left for evaporation of excess dimethylamine. The reaction mixture was concentrated in vacuo, the residue was dissolved in ethyl acetate and was extracted with 1N-sodium hydroxide solution and water. After drying with sodium sulfate the organic layer was evaporated and the residue was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene as ale is new. Thus obtained amide C-130 was isolated and optionally purified by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane.

Example X-26. Obtaining the compounds of formula:

To a suspension of Aenon C-114 (3.5 g, 10 mmol) in utilitiarian (10 ml) and anhydrous ethanol (10 ml) was added monohydrate p-toluensulfonate acid (0.05 g). The reaction mixture was stirred for 30 minutes at room temperature and was suppressed by adding a few drops of pyridine. After stirring for 5 minutes at 0°the precipitate was filtered, washed with a small amount of methanol and recrystallized from ethanol or ethyl acetate and hexane containing a trace amount of pyridine, resulting in a net simple enol ether C-131. Alternatively, after adding pyridine this reaction mixture can be treated by removing all solvent in vacuo and crystallization of the residue by adding solvents such as hexane, simple ether, ethyl acetate or hexane. Raw simple enol ether C-131 recrystallized as described above.

Example X-27. Receive:

To a cooled (-78° (C) to a solution of bis(trimethylsilyl)amide lithium (10 ml of 1.0 M solution in tetrahydrofuran) dropwise over 20 minutes add the Yali simple solution of enol ether C-131 (3.8 g, 10 mmol) in tetrahydrofuran (20 ml). The reaction mixture was stirred at -78°C for 10 minutes and the solution was added fenilcetonuria (1.9 g, 10 mmol) in tetrahydrofuran (5 ml). The reaction mixture was stirred for 5 minutes and extinguished by adding a 1% solution of sodium bisulfate. Then the reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers washed with 5% aqueous sodium bicarbonate solution and water and dried with sodium sulfate. The drying agent was filtered and the filtrate was concentrated in vacuum. The residue was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents and received net selenide-132.

Example X-28. Receive:

To a cooled (0° (C) to a solution of selenide-132 (g, 10 mmol) and pyridine (I,61 ml, 20 mmol) in methylene chloride (40 ml) was slowly added a solution of hydrogen peroxide (3.1 g of 30% solution, 27 mmol) in water (3 ml). The temperature was maintained at less than 30-35°C. After reduction ekzotermicheskie water bath was removed and the reaction mixture was intensively stirred for 15 minutes at room temperature. Reational mixture was diluted with methylene chloride and washed with 5% sodium bicarbonate solution and water and dried with sodium sulfate. Dewatering agent Phil is trevali and the filtrate was concentrated in vacuum. The residue was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene or ethyl acetate and hexane as eluents and received net unsaturated ketone With a-133, which is then purified by recrystallization from ethyl acetate and hexane or alcohol.

Example X-29. Receive:

A solution of the ketone With a-133 (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 vacuum. 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 obtain the crude ketone-134. This crude product was purified by chromatography on silica gel using mixtures of ethyl acetate and toluene as eluents and received net ketone-134, which was further purified by recrystallization from ethyl acetate and hexane or alcohol.

1. A method of obtaining a 3-keto-7α-alkoxycarbonylmethyl Δ4,5-steroid of General formula

whereselected fromand

R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R21represents hydrogen or alkyl;

R26represents a C1-C4alkyl;

R8and R9taken together form a heterocyclic ring system,

including interaction alkylating agent with 4,5-dihydro-5,7-lakestream substrate of General formula:

where R18represents a C1-C4alkyl, or R18About groups taken together form an O,O-oxyalkylene bridge or ketogroup, and, R3, R8and R9have the meanings specified above, in the presence of a base.

2. The method according to claim 1, where the specified steroid substrate and the specified steroid product substituted in the 17-position spirolactone Deputy corresponding to formula XXXIII

3. The method according to claim 1, where the specified lactone substrate contains 3 dialkoxy deputies.

4. The method according to claim 1, where the specified 5,7-lactone is subjected to interaction with alkylhalogenide in the presence of a base.

5. The method of obtaining 4,5-dihydro-5,7-lactonitrile derivative of the formula

where , R3, R8, R9and R18matter where specified in claim 1, consisting in converting the corresponding 7-lanzamientos steroid 7-carboxyphenyl steroid and subsequent conversion of 7-carboxylester steroid in the corresponding 5,7-lactosamine steroid.

6. The method according to claim 5, where Δ-4,5-7-carboxyamide the steroid group OR18taken together, they form a 3-ketogroup that transform with getting catalogo intermediate compounds containing 3-dialkoxy-5,7-lactone, and the specified 3-dialkoxy-5,7-lactone is then hydrolized under acidic conditions to produce 3-keto-5,7-lactone.

7. The method of obtaining the compounds corresponding to formula II

,

where R1represents an alpha-oriented C1-C4alkoxycarbonyl radical;

R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R8and R9taken together form a heterocyclic ring system;

which includes alkylation of compounds of formula EI by hydrolysis with subsequent interaction with the alkylating reagent in the presence of a base, where the compound of the formula EI has the structure:

,

where R3, R8and R9 have the values specified above;

R17represents a C1-C4alkyl.

8. The method of obtaining the compounds of formula IIC

,

where R1represents an alpha-oriented C1-C4alkoxycarbonyl or hydroxycarbonyl radical;

R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

which includes alkylation of compounds of General formula E by hydrolysis with subsequent interaction with the alkylating reagent in the presence of a base, where the compound of formula E has the structure

,

where R3has the values listed above;

R17represents a C1-C4alkyl.

9. The method of obtaining the compounds of formula E

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R17represents a C1-C4alkyl;

which includes thermal decomposition of compounds corresponding to the formula DE2, in the presence of alkali metal halide, where the aforementioned compound of the formula DE2 has the structure

,

where R12is the th 1-C4alkyl;

R3and R17have the values specified above.

10. The method of obtaining the compounds of formula DE2

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R12and R17represent1-C4alkyl;

which involves the condensation of the compounds of formula DE1 with diallylmalonate in the presence of a base, where the aforementioned compound of the formula DE1 has the structure:

,

where R3and R17have the values specified above.

11. The method of obtaining the compounds of formula DE1

,

where R3selected from the group comprising hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R17represents a C1-C4alkyl;

which includes the interaction of the compounds of formula D with ridom sulfone in the presence of a base, where the aforementioned compound of formula D has the structure:

,

where R3and R17have the values specified above.

12. The method of obtaining the compounds of formula D

,

where R3represents hydrogen, halogen, lower alkyl, lower al is hydroxy or cyano;

R17represents a C1-C4alkyl;

which includes the hydrolysis of compounds of formula C With formation of 7α-carboxylic acid and carrying out the reaction in acidic conditions with trialkylaluminium, where the compound of formula has the structure

,

where R3has the values listed above.

13. The method of obtaining the compounds of formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R26represents a C1-C4alkyl;

R80and R90independently selected from R8and R9respectively, or R80and R90together form ketogroup;

which includes alkylation of compounds of formula I by reacting with an alkylating reagent in the presence of a base, where the compound of formula I has the structure

,

where R3, R80and R90have the values specified above.

14. The method of obtaining the compounds of formula IE

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R26represents a C1-C4alkyl;

which includes alkylation of compounds of General formula 211 by reacting with an alkylating reagent in the presence of a base, where the compound of formula 211 has a structure

,

where R3has the values listed above.

15. The method of obtaining the compounds of formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R80and R90independently selected from R8and R9respectively, or R80and R90together form ketogroup;

R8and R9independently represent hydroxycarbonyl, or R8and R9together represent a heterocyclic ring structure;

which involves the oxidation of compounds of formula I, where the compound of formula I has the structure

,

where R3, R80and R90have the values specified above.

16. The method according to clause 15, where R80and R90together with(17) denote

where X represents oxo;

Y1represents hydroxy;

Y2represents hydroxy or lower alkoxy.

17. The method according to clause 15, where R80and R90 taken together with(17), mean

18. The method of obtaining compounds of General formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R8and R9independently represent hydroxycarbonyl, or R8and R9taken together, represent a heterocyclic ring structure;

which involves reacting an intermediate of 3-keto-5,7-polyacetale formula AS peroxide oxidizer, where the aforementioned compound of the formula AS corresponds to the formula

where R3, R8and R9have the values specified above.

19. The method according to p, where R8and R9taken together with(17), mean

where X represents oxo;

Y1represents hydroxy;

Y2represents hydroxy or lower alkoxy.

20. The method according to p, where R8and R9taken together with(17), mean

21. The method of obtaining compounds of General formula I

,

where R3represents hydrogen, halogen, lower alkyl or the Chille alkoxy or cyano;

R80and R90independently selected from R8and R9respectively, or R80and R90together form ketogroup;

R8and R9independently represent hydroxycarbonyl, or R8and R9together represent a heterocyclic ring structure;

which involves reacting an intermediate of 3-keto-5,7-polyacetale formula AS peroxide oxidizer, where the aforementioned compound of the formula AS corresponds to the formula

where R3, R8and R9have the values specified above.

22. The method according to item 21, where R8and R9taken together with(17), mean

where X represents oxo;

Y1represents hydroxy;

Y2represents hydroxy or lower alkoxy.

23. The method according to item 21, where R8and R9taken together with(17), mean

24. The method of obtaining the compounds corresponding to the formula I,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R80and R90independently selected from R8and R9respectively, or R80and R90

R8and R9independently represent hydroxycarbonylmethyl or R8and R9together represent a heterocyclic ring structure;

-E-e - is selected from the group including

and

where R21represents hydrogen or alkyl;

which includes the hydrolysis of compounds of General formula A208

,

where-e-E-, R3, R80and R90have the values specified above;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge; R20represents a C1-C4alkyl.

25. The method of obtaining compounds of General formula A;

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

R20represents a C1-C4alkyl;

-E-e - is selected from the group including

and

where R21independently selected from hydrogen and alkyl;

which involves reacting compounds of General formula I with the lower alcohol and acid, where the aforementioned compound of the formula I has the structure

,

where-e-E-, R3and R19have the values specified above.

26. The method of obtaining the compounds of formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

-E-e - is selected from the group including

,

and

where R21independently selected from hydrogen and alkyl;

which includes the hydrolysis of compounds of General formula I, where the aforementioned compound of the formula I has the formula

where-o-o - and R3have the values specified above;

R18represents a C1-C4alkyl, or R18About groups taken together form an O,O-oxyalkylene the bridge.

27. The method of obtaining joint the formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

-E-e - is selected from the group including

and

where R21independently selected from hydrogen and alkyl;

which includes the protection of closability the compounds of formula A by interacting with alkanols in acidic conditions in the presence of orthoformiate, where the aforementioned compound of the formula I has the structure

,

where-o-o - and R3have the values specified above;

obtaining, thus, intermediate simple ester 3-enol of formula I

,

where-o-o - and R3have the values specified above;

R18represents a C1-C4alkyl, or R18About groups taken together form an O,O-oxyalkylene bridge;

the recovery of the compounds of formula A with obtaining the compounds of formula I:

,

where-e-E-, R3 and R18have the values specified above;

the hydrolysis of the compounds of formula A obtaining thus the compounds of formula A.

28. The method of obtaining the compounds of formula I:

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R18represents a C1-C4alkyl, or R18Oh-group at C-17, taken together, form 0,0-oxyalkylene bridge;

-E-e - is selected from the group including

and

where R21independently selected from hydrogen and alkyl;

which includes the recovery of the compounds of formula I:

where-e-E-, R3and R18have the values specified above.

29. The method according to any of PP-28, where R3represents hydrogen.

30. The method according to any of PP-28, where R3represents hydrogen and-o-o - is selected from

and

31. The method of obtaining epoxysilane, comprising contacting the substrate compounds having olefinic double bond with a peroxide compound in risotti peroxide activator, where specified peroxide activator is chlorodifluoroacetate or corresponds to the compound of formula

,

where

Rpselected from the group including, alkenyl, quinil and(CX4X5)n-;

X1X2X3X4and X5independently selected from halogen, hydrogen, alkyl, halogenoalkane, cyano and zainoulline;

n is 0, 1 or 2;

provided that when n is 0, then at least one of X1X2and X3represents halogen,

when Rprepresents -(CX4X5)nand n is 1 or 2, then at least one of X4and X5represents a halogen.

32. The method according to p, where n is 0 and at least two of X1X2and X3represent halogen or perhalogenated.

33. The method according to p, where all X1X2X3X4and X5represent halogen or perhalogenated.

34. The method according to p where specified peroxide activator selected from the group comprising chlorodifluoroacetate and heptafluorobutyrate.

35. The method according to p, where the aforementioned substrate compound has the formula II

,

where R3represents hydrogen, halogen, lower alkyl, lower and is cocci or cyano;

R1represents an alpha-oriented C1-4alkoxycarbonyl radical;

R8and R9independently selected from hydroxycarbonyl; or R8and R9taken together form a heterocyclic ring structure.

36. The method according to p, where R3represents hydrogen.

37. The method according to p, where the aforementioned substrate compound selected from the group including

and the reaction product of epoxidation selected from the group including

38. The method according to p, where the aforementioned substrate compound selected from the group including

and the reaction product of epoxidation selected from the group including

39. The compound of formula D

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R17represents a C1-C4alkyl.

40. The compound of formula E

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R17represents a C1-C4alkyl.

41. The compound of formula F

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy and cyano.

42. The compound of formula 211

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy and cyano.

43. The compound of formula I

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R80and R90independently selected from R8and R9, respectively, or R80and R90together form ketogroup;

R8and R9independently selected from hydroxycarbonyl; or R8and R9taken together form a heterocyclic ring structure.

44. The compound of formula I

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R80and R90independently selected from R8and R9respectively, or R80and R90together form ketogroup;

R8and R9independently selected from hydroxycarbonyl, or R8and R9taken together form a heterocyclic ring structure;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

45. The compound of formula 208:

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

R20represents a C1-C4alkyl;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

46. The compound of formula 207

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

R20represents a C1-C4alkyl;

R25represents a C1-C4alkyl;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

47. The compound of formula I

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

R20represents a C1-C4alkyl;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

48. The compound of formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

R20represents a C1-C4alkyl;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

49. The compound of formula I

,

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R19represents a C1-C4alkyl, or R19O-groups taken together form an O,O-oxyalkylene bridge;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

50. The compound of formula I

where R3represents hydrogen, halogen, lower alkyl, lower alkoxy or cyano;

R18represents a C1-C4alkyl, or R18O-group at C-17, taken together, form O,O-oxyalkylene bridge;

-E-e - is selected from

and

where R21represents hydrogen or alkyl.

51. Connection item 50 formula A, where R3represents hydrogen.

52. The compound according to any one of paragraphs 44 to 51, where R3represents hydrogen;

-E-e - is selected from

and

where R21represents hydrogen.

53. The compound according to any one of paragraphs 44 to 51, where R3represents hydrogen;

-E-e - is a

where R21represents hydrogen.

54. The compound of formula 303

where R1represents an alpha-oriented C1-C4alkoxycarbonyl or hydroxycarbonyl radical;

R8and R9independently represent hydroxycarbonyl, or R8and R9together represent a heterocyclic ring structure.

55. Connection by item 54 of the formula I

where R1represents an alpha-oriented C1-C4alkoxycarbonyl or hydroxycarbonyl radical;

X represents oxo;

Y1is a guide is hydroxy;

Y2represents hydroxy or lower alkoxy.

56. Connection § 55 of the formula

Priority items:

11.12.1996 according to claims 1 to 30, 39-56;

11.12.1997 on p-38.



 

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SUBSTANCE: method for enzymatic conversion of phytosterol compositions to androstenedion (androst-4-ene-3,17-lion, AD) and androstadienedion (androsta-1,4-diene-3,17-dion, ADD) includes heating of phytosterol composition in presence of one or more solubilizers up to 100-130°C to form paste-like solution. Solubilizers are selected from polypropylene glycol, silicone or vegetable oil. Prepared paste-like mass is charged into bioreactor containing microorganism Mycobacterium MB 3683 and inorganic salt medium followed byfermentation. Method of present invention makes it possible to sufficiently increase of phytosterol composition concentration (30 g or more for 1 l of broth) and to increase produced product yield up to 80-90 %.

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SUBSTANCE: invention relates to a method for preparing derivatives of steroid glycosides from the plant Ruscus aculeatus (ruscosaponins). Method for preparing desglucodesrhamnoruscin involves hydrolysis of steroid glycosides from Ruscus aculeatus (ruscosaponins) by fermentation of substrate containing indicated glycosides using fungus of species Aspergillus niger. Method provides preparing desglucodesrhamnoruscin with the high degree of effectiveness.

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2 cl, 1 ex

FIELD: organic chemistry, chemical technology, pharmacy.

SUBSTANCE: invention describes the improved method for preparing flumetasone (6α,9α-difluoro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), flumetazone 21-acetate or its 17-carboxyl-androstene analogue of the formula (I) . Method involves interaction of benzoyl chloride with compound of the formula (II) in pyridine medium of its mixture with N,N'-dimethylacetamide to prepare 3-enol ester of the formula (IIIa) and it's the following interaction with 1-(chloromethyl)-4-fluoro-1,4-diazonium-bicyclo[2.2.2]octane-bis(tetrafluoroborate) in acetonitrile medium and water to prepare compound of the formula (IIIb) and the following removing the protective group in compound of the formula (IIIb) at the position C3 in medium of aqueous metabisulfite and ammonia to prepare compound of the formula (IV) . After the fluorination reaction of 9,11-epoxy group in compound of the formula (IV) using HF flumetasone 21-acetate is prepared followed by the selective hydrolysis with KOH in methanol (CH3OH) medium in the presence or absence of H2O2 to prepare compound of the formula (I) or flumetasone, respectively.

EFFECT: improved preparing method.

3 cl, 5 ex

FIELD: organic chemistry, steroids, pharmacy.

SUBSTANCE: invention relates to a new type of selective estrogens comprising steroid structure of the general formula (I) with nonaromatic ring A and free of bound hydroxyl group at carbon atom 3 wherein R1 means hydrogen atom (H), (C1-C3)-alkyl or (C2-C3)-acyl; R2 means hydrogen atom (H), α-(C1-C4)-alkyl, α-(C2-C4)-alkenyl or α-(C2-C4)-alkynyl; R3 means hydrogen atom (H) or (C1-C4)-alkyl at position 16 of steroid structure; R4 means ethynyl; R5 means hydrogen atom (H), (C1-C3)-alkyl or (C2-C3)-acyl; R6 means (C1-C5)-alkyl, (C2-C5)-alkenyl, (C2-C5)-alkynyl being each of that is substituted optionally with chlorine or fluorine atom; dotted line means the optional double bond. Compounds of the formula (I) elicit the selective affinity to ERα-receptors.

EFFECT: valuable properties of compounds and composition.

4 cl, 3 sch, 1 tbl, 8 ex

The invention relates to an improved method of obtaining carboxamido-4-azasteroid General formula I, in which the dashed lines independently represent either simple or double bond, R, R1, R2and R3each represents hydrogen or an organic radical comprising processing the corresponding intermediate compounds 17-carbonyl-imidazoles anhydrous acid in the presence of amine and, optionally, hydrogenation of the compounds obtained

The invention relates to an improved method of isolation and purification of saponins

The invention relates to 14,17-C2-bridged steroids of formula I, where R3- O, R6- Hor-(C1-C4)-alkyl, where R6and R7together form an additional bond; R7-or-(C1-C4)-alkyl, where R6and R6both H, or R9and R10each H or together form a bond, R11and R12each H or together form a bond, R13- CH3or2H5; R15- H or C1-C3-alkyl; R16and R16independently H, (C1-C3)-alkyl or C1-C4alkenyl or together form a (C1-C3-alkyliden; R15and R16together form a cyclewhere n = 1, and h - O and R16- N- H, (C1-C3)-alkyl,- H, (C1-C3)-alkyl,- H, (C1-C3)-alkyl or HE; except 14,17-ethano-19-norpregna-4-ene-3,20-dione

The invention relates to compounds that perform new functions inhibitors of bone ratably/promoters osteogenesis
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