KR100506007B1 - Processes for preparation of 9,11-epoxy steroids and intermediates useful therein - Google Patents

Abstract

Multiple novel reaction schemes, new process steps and new intermediates may be used for

Formula I

(Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group,

R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano and aryloxy groups;

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

-BB- is a -CHR ⁶ -CHR ⁷ -group or an alpha- or beta-oriented group

Formula III

Wherein R ⁶ and R ⁷ are independently selected from hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy );

R ⁸ and R ⁹ are independently a group consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or together with R ⁶ or R ⁷ , a carbocyclic or heterocyclic type fused to a 5-membered cyclic D ring Constituting a ring structure).

Description

PROCESSES FOR PREPARATION OF 9,11-EPOXY STEROIDS AND INTERMEDIATES USEFUL THEREIN

The present invention provides a novel process for preparing 9,11-epoxy steroid compounds, especially the 20-spiroxane series and analogs thereof, novel intermediates useful for preparing steroid compounds, and methods for preparing such novel intermediates. It is about. More specifically, the present invention relates to methyl hydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone (eplerenone or epoxymex And a new and advantageous method for the production).

Methods for preparing 20-spiroxane series compounds are described in US Pat. No. 4,559,332. Compounds prepared according to the method of U.S. Patent No. 4,559,332 have ring E containing open oxygen of the general formula:

(Wherein -AA- represents a -CH ₂ -CH ₂ -or -CH = CH- group;

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

-BB- is a -CH ₂ -CH ₂ -group or the following group III of the α- or β-orientation:

In which R ⁶ and R ⁷ are hydrogen;

X represents two hydrogen atoms or oxo groups;

Y ¹ and Y ² together represent an oxygen bridge -O- or Y ¹ represents hydroxy and Y ² represents hydroxy, lower alkoxy or a lower alkanoyloxy group if X represents H _2. )

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

U.S. Patent No. 4,559,332 describes many methods for preparing epoxymexrenone and related compounds of Formula IA above. The new use of epoxymexrenone for a wider range of clinical uses necessitates improved methods of preparing these and other related steroids.

1 is a schematic flow chart showing a method of converting a canrenon or canrenone derivative into a corresponding 11α-hydroxy compound in vivo.

2 is a schematic flowchart of a preferred method for in vivo conversion / 11-α-hydroxylation of canrenones and canrenone derivatives.

3 is a schematic flowchart of a particularly preferred method for in vivo conversion / 11-α-hydroxylation of canrenones and canrenone derivatives.

FIG. 4 shows particle size distribution for canrenones prepared according to the method of FIG. 2.

FIG. 5 shows particle size distribution for canrenone sterilized in a conversion fermenter according to the method of FIG. 3.

Corresponding reference symbols indicate corresponding parts throughout the drawings.

Description of Preferred Embodiments

According to the present invention, a variety of novel process methods have been devised for the preparation of epoxymexrenone and other compounds according to formula I:

Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group;

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

R ¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxyalkyl group;

-BB- is a group -CHR ⁶ -CHR ⁷ -or alpha- or beta-oriented of the following group III:

Formula III

Wherein R ⁶ and R ⁷ are independently hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Selected from the group);

R ⁸ and R ⁹ are independently a group consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ are fused to a 5-membered cyclic D ring It constitutes a borosilicate or heterocyclic ring structure.

Unless stated otherwise, the organic groups referred to in the present specification as "lower" contain at least 7, preferably 1 to 4 carbon atoms.

Lower alkoxycarbonyl groups are preferably alkyl groups having 1 to 4 carbon atoms, such as those derived from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl groups; Especially preferred are methoxycarbonyl, ethoxycarbonyl and isopropoxycarbonyl groups. Lower alkoxy groups are preferably derived from one of the aforementioned C ₁ -C ₄ alkyl groups, in particular derived from primary C ₁ -C ₄ alkyl groups, with particular preference being given to methoxy groups. Lower alkanoyl groups are preferably derived from straight chain alkyl having 1 to 7 carbon atoms, with particular preference being to formyl and acetyl groups.

The methylene bridge between the 15- and 16-positions is preferably of the β-orientation.

A preferred class of compounds that can be prepared according to the methods of the present invention are the 20-spiroxane compounds described in US Pat. No. 4,559,332, which correspond to formula IA:

Formula IA

Where

-AA- represents a -CH ₂ -CH ₂ -or -CH = CH- group;

-BB- represents the formula IIIA of the -CH ₂ -CH ₂ -group or alpha- or beta-alignment:

R ¹ represents an alpha-oriented lower alkoxycarbonyl group or hydroxycarbonyl group;

X represents two hydrogen atoms, oxo or = S;

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

Y ¹ represents a hydroxy group and Y ² represents a hydroxy, lower alkoxy group, or if X represents H ₂ , it also represents a lower alkanoyloxy group.

Preferred are the 20-spiroxane compounds prepared according to the novel process of the invention, compounds of formula I in which Y ¹ and Y ² together represent an oxygen bridge -O-.

Particularly preferred compounds of formula I are those of formula I in which X represents an oxo group. Among the 20-spiroxane compounds of the formula (IA) in which X represents an oxo group, most preferred are those in which Y ¹ represents oxygen bridge -O- together with Y ² .

As mentioned previously, 17β-hydroxy-21-carboxylic acid may also be present in its salt form. Especially from metal and ammonium salts, such as alkali and alkaline earth metal salts, for example sodium, calcium, magnesium and, preferably, potassium salts, and ammonia or derived from suitable, preferably physiologically acceptable organic nitrogen-containing bases. Ammonium salts can be considered. Bases include amines such as lower alkylamines (such as triethylamine), hydroxy-lower alkylamines (such as 2-hydroxyethylamine, di- (2-hydroxyethyl) -amine or tri- (2-hydroxy). Oxyethyl) -amines), cycloalkylamines (such as dicyclohexylamine) or benzylamines (such as benzylamine and N, N'-dibenzylethylenediamine), as well as nitrogen-containing heterocyclic compounds such as aromatics Nitrogen-containing heterocyclic compounds having properties (such as pyridine or quinoline) or nitrogen-containing heterocyclic compounds having at least partially saturated heterocyclic rings (such as N-ethylpiperidine, morpholine, pipepe Lazine or N, N'-dimethylpiperazine) are contemplated.

Further preferred compounds are alkali metal salts, in particular potassium salts, of compounds of formula IA in which R ¹ represents alkoxycarbonyl, X represents oxo, and each of Y ¹ and Y ² represents hydroxy.

Particularly preferred compounds of formulas I and IA are, for example:

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

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

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

1,2-dehydrated analogs of each of the 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-bismethylene-20-spirox-4-ene-3,21-dione, and

1,2-dehydrated analogs of each of the compounds;

9α, 11α-epoxy-7α-methoxycarbonyl-17β-hydroxy-3-oxo-pregin-4-ene-21-carboxylic acid,

9α, 11α-epoxy-7α-ethoxycarbonyl-17β-hydroxy-3-oxo-pregin-4-ene-21-carboxylic acid,

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

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

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

9α, 11α-epoxy-17β-hydroxy-6β, 7β; 15β, 16β-bismethylene-3-oxo-pregan-4-ene-21-carboxylic acid, and

Alkali metal salts of each of these acids, in particular potassium salts or ammonium, and corresponding 1,2-dehydration analogs of each or its salts of the carboxylic acids mentioned;

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

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

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

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

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

9α, 11α-epoxy-17β-hydroxy-17α- (3-hydroxy-propyl) -3-oxo-androst-4-ene-7α-carboxylic acid methyl ester, ethyl ester and isopropyl ester,

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

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

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

17α- (3-acetoxypropyl) and 17α- (3-formyloxypropyl) analogs of the androstane compounds mentioned above, and androst-4-en-3-one and 20-spirox-4-ene- 1,2-dehydration analogs of all mentioned compounds of the 3-one series.

The chemical names of the compounds of formulas I and IA, and analogous compounds having the same structural characteristics, are derived according to the currently accepted nomenclature in the following way: For compounds wherein Y ¹ represents -O- with Y ² , 20-spiroxane (eg, a compound of Formula IA wherein X represents oxo and Y ¹ represents -O- with Y ² is derived from 20-spiroxane-21-one); For compounds wherein each of Y ¹ and Y ² represents hydroxy and X represents oxo is selected from 17β-hydroxy-17α-pregnene-21-carboxylic acid; And for compounds in which each of Y ¹ and Y ² represents hydroxy and X represents two hydrogen atoms is named 17β-hydroxy-17α- (3-hydroxypropyl) -androstane. Since the cyclic and open chain forms, ie lactone and 17β-hydroxy-21-carboxylic acid and salts thereof, are each so closely related to each other that the latter are simply regarded as the former, dehydrated form, It is to be understood that all of the final products of the formula I and the starting materials and the intermediates of similar structures are present in each case together, unless specifically stated otherwise.

According to the invention, several separate preparation methods have been devised for the preparation of the compounds of formula I in high yields and at reasonable costs. Each of the methods of synthesis proceeds through the preparation of a series of intermediates. Many of these intermediates are novel compounds, and methods of making these intermediates are also novel methods.

Method 1: Start with Canrenone or Related Substance

One preferred reaction method for preparing compounds of formula I begins with canrenone or related starting materials corresponding to formula XIII below (or alternatively, the preparation method can be started using androstenedione or related starting materials). have):

Where

-AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group;

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

-BB- is a group -CHR ⁶ -CHR ⁷ -or an alpha- or beta-oriented group of:

Formula III

Wherein R ⁶ and R ⁷ are independently hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Selected from the group);

R ⁸ and R ⁹ are independently a group consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Or R ⁸ and R ⁹ together form a keto, carbocyclic or heterocyclic ring structure, or R ⁸ and R ⁹ together with R ⁶ or R ⁷ are fused to a 5-membered cyclic D ring A carbocyclic or heterocyclic ring structure.

Using biotransformation methods of the type shown in FIGS. 1 and 2, an α-oriented 11-hydroxy group is introduced into a compound of formula XIII, thereby preparing a compound of formula VIII:

Formula VIII

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined for formula XIII. Preferably, the compound of formula XIII is of the structure

Preferably, the 11α-hydroxy product is represented by the formula:

Formula VIIIA

And having salts of compounds in which X represents oxo and Y ² represents hydroxy:

In each of the above formulas,

-AA- represents a -CH ₂ -CH ₂ -or -CH = CH- group;

-BB- is a -CH ₂ -CH ₂ -group or an alpha- or beta-oriented group of:

Formula IIIA

Represents;

R ³ is hydrogen, lower alkyl or lower alkoxy;

X represents two hydrogen atoms, oxo or = S;

Y ¹ and Y ² together represent an oxygen bridge —O—, or Y ¹ represents hydroxy, Y ² represents hydroxy, lower alkoxy, or if X represents H _2, then lower alkane Noyloxy.

More preferably, the compounds of formula VIIIA produced in the reaction are -AA- and -BB-, respectively -CH ₂ -CH _2- ; R ³ is hydrogen; Y ¹ , Y ² and X are as defined for Formula XIIIA; Corresponding to compounds of formula VIIIA when R ⁸ and R ⁹ together form the following 20-spiroxane structure:

(Formula XXXIII)

Preferred organisms that can be used in this hydroxylation step include Aspergillus ochraceus NRRL 405, Aspergillus okraceus ATCC 18500, Aspergillus niger ATCC 16888 and ATCC 26693, A. nidulans ATCC 11267, Rhizopus oryzae ATCC 11145, R. stolonifer ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillus megaterium ATCC 14945, Pseudomonas cruciviae ATCC 13262, and Tricothecium roseum ATCC 12543. Other preferred organisms include Fusarium oxysporum f. Sp. Cepae ATCC 11171 and R. arrhizus ATCC 11145.

Other organisms that are active against this reaction include Absidia coerula ATCC 6647, A. glauca ATCC 22752, Actinomucor elegans ATCC 6476, Aspergillus. A. flavipes ATCC 1030, A. fumigatus ATCC 26934, Beauveria bassiana ATCC 7159 and ATCC 13144, Botryosphaeria obtusa ) IMI 038560, Calonectria decora ATCC 14767, Chaetomium cochliodes ATCC 10195, Corynectria cassiicola ATCC 16718, Cunninghamella Blaques Les Ana ( Cunninghamella) blakesleeana ) ATCC 8688a, C. echinulata ATCC 3655, C. elegans ATCC 9245, Curvularia clavata A TCC 22921, C. lunata ATCC 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 ( M griseo-cyanus ) ATCC 1207A, Myrothecium verrucaria ATCC 9095, Nocardia corallina ATCC 19070, Paecilomyces carneus ATCC 46579, Penicillum par Tulum (Penicillum patulum) ATCC 24550, phyto Mai access to art-Oliva three mouse (Pithomyces atro-olivaceus) IFO 6651 , Phyto My process Sino money tooth (P. cynodontis) ATCC 26510, peak no sports Solarium species (P ycnosporium sp. ) ATCC 12231, Saccharopolyspora erythrae ATCC 11635, Sepedonium chrysospermum ATCC 13378, Stachylidium bicolor ATCC 12672, Streptomyces hygroscopius ( S. hygroscopicus ) ATCC 27438, S. purpurascens ATCC 25489, Syncephalastrum racemosum ATCC 18192, Thamnostylum piriforme ATCC 8992, Tierravia Thielavia terricola ATCC 13807, and Verticillium theobromae ATCC 12474.

Additional organisms that are expected to be active against 11α-hydroxylation include Cephalosporium aphidicola [Phytochemistry (1996), 42 (2), 411-415], Cocliobolus lunatas ( Cochliobolus lunatas ) [J. Biotechnol. (1995), 42 (2), 145-150, Tieghemella orchidis [Khim.-Farm. Zh. (1986), 20 (7), 871-876, T. hyalospora [Khim.-Farm. Zh. (1986), 20 (7), 871-876], Monosporium olivaceum [Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110], Aspergillus Ustus [Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110], Fusarium graminearum [Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110], Verticillium glaucum [Acta Microbiol. Pol., Ser. B. (1973), 5 (2), 103-110, and R. nigricans [J. Steroid Biochem. (1987), 28 (2), 197-201.

11β-hydroxy derivatives of androstenedione and mexrenone can be prepared according to the in vivo conversion method described in Examples 19A and 19B, respectively. The inventors have inferred that the corresponding β-hydroxy isomers of the compounds of formula VIII having C11β-hydroxy substituents instead of C11α-hydroxy substituents are also suitable microorganisms, such as those capable of performing 11β-hydroxylation. It is assumed that it can be prepared using similar in vivo conversion methods using one or more microorganisms described herein.

In preparation for fermentation at the manufacturing scale for the hydroxylation of canrenone or other substrates of formula XIII, a seed fermentation system in which the cell inoculum contains a seed fermenter, or a series of two or more seed fermenters Is manufactured in. A working stock spore suspension is introduced with nutrient solution for cell growth in the first seed fermenter. If the inoculum volume required or required for preparation exceeds that produced in the first seed fermenter, the inoculum volume can be amplified gradually and geometrically by running through the fermentor remaining in the seed fermentation train. Preferably, the inoculum prepared in the seed fermentation system is of sufficient volume and is viable cells to achieve rapid onset of reaction, relatively short production batch cycle, and high production fermenter activity in the production fermenter. Whatever the number of vessels in the train of the seed fermenter, the second and subsequent seed fermentors are preferably sized such that the degree of dilution at each stage of the train is essentially the same. The initial dilution rate of the inoculum in each seed fermenter may be approximately equal to the dilution rate in the production fermenter. Canrenone or other substrates of formula XIII are packed together with the inoculum and the nutrient solution into the preparation fermenter, where the hydroxylation reaction is carried out.

Spore suspensions filled into the seed fermentation system were filled from vials of working stock spore suspensions taken from a plurality of vials consisting of working stock cell banks stored under cryogenic conditions prior to use. The working stock cell bank is subsequently derived from a master stock cell bank prepared in the following manner. Spore samples obtained from a suitable source such as ATCC are first suspended in an aqueous medium such as saline, nutrient solution or surfactant solution (such as nonionic surfactant such as Tween 20 at a concentration of about 0.001% by weight) and in a culture plate Distributing the suspension causes the spores to multiply in the plate. The culture plate then typically contains a solid nutrient mixture based on non-digestible polysaccharides such as sugars. The solid nutrient mixture preferably comprises between about 0.5% by weight and about 5% by weight glucose, between about 0.05% by weight and about 5% by weight nitrogen sources such as peptone, between about 0.05% by weight and about 0.5% by weight phosphorus, Alkali metal phosphates such as ammonium or dipotassium hydrogen phosphate, yeast lysates or extracts (or other amino acid sources such as meat extracts or brain-heart fusions), about 1 weight, between about 0.25 weight% and about 2.5 weight% Agar or other non-digestible polysaccharides are contained between% and about 2% by weight. Optionally, the solid nutrient mixture may further comprise and / or contain between about 0.1 wt% and about 5 wt% malt extract. The pH of the solid nutrient mixture is preferably between about 5.0 and about 7.0 and is adjusted by alkali metal hydroxide or orthophosphoric acid as necessary. Useful solid growth media include:

1.Solid Medium # 1: 1% Glucose, 0.25% Yeast Extract, 0.3% K ₂ HPO ₄ , and 2% Agar (Bacto); pH was adjusted to 6.5 with 20% NaOH.

2. Solid Medium # 2: 2% Peptone (Bacto), 1% Yeast Extract (Bacto), 2% Glucose, and 2% Bacto; PH was adjusted to 5 with 10% H ₃ PO ₄ .

3. Solid Medium # 3: 0.1% Peptone (Bacto), 2% Malt Extract (Bacto), 2% Glucose, and 2% Bacto; pH is 5.3.

4. Liquid medium: 5% waste molasses, 0.5% corn steep liquid, 0.25% glucose, 0.25% NaCl, and 0.5% KH ₂ PO ₄ ; pH adjusted to 5.8.

5. Difco fungus agar (low pH).

The number of agar plates used in the development of the master stock cell bank can be selected according to future needs for the master stock, but typically about 15 to about 30 plates are selected. After an appropriate growth period, such as between 7 and 10 days, the plates are scraped for harvesting of spores in the presence of an aqueous excipient, typically saline or buffer, and the resulting master stock suspension is prepared in multiples of 1.5 ml. Each small vial of is contained in a volume of, for example, 1 ml. In order to prepare working stock spore suspensions for use in the study or preparation of fermentation operations, the contents of one or more of these second generation master stock vials are distributed in agar plates and described above to prepare master stock spore suspensions. Can be incubated on the agar plate in a manner. Given the basic manufacturing operation, as many as 100 to 400 plates can be used to make the second generation working stock. Each plate is scraped into a separate working stock vial, where each vial typically contains 1 ml of the already prepared inoculum. For permanent preservation, both the master stock suspension and the second generation production inoculum are advantageously stored in a vacuum chamber of cryogenic storage vessels containing liquid nitrogen or other cryogenic liquids.

In the method illustrated in FIG. 1, an aqueous growth medium is prepared comprising a nitrogen source such as peptone, a yeast derivative or equivalent, a phosphorus source such as glucose, and phosphate. Spores of microorganisms are cultured in this growth medium in the seed fermentation system. Preferred microorganisms are Aspergillus okraceus NRRL 405 (ATCC 18500). The seed stock thus prepared is then introduced into the resulting fermentor together with the substrate of formula XIII. The fermentation broth is stirred and vented for a time sufficient to allow the reaction to proceed to the desired degree.

The medium for the seed fermenter preferably includes an aqueous mixture containing the following: between about 0.5% by weight and about 5% by weight glucose, between about 0.05% by weight and about 5% by weight nitrogen source, such as peptone A source of phosphorus between about 0.05% and about 0.5% by weight such as ammonium or alkali metal phosphate such as monobasic ammonium phosphate or dipotassium hydrogen phosphate, yeast lysate between about 0.25% and about 2.5% by weight Or an extract (or other amino acid source, such as, for example, a soluble substance of a still), agar or other non-digestible polysaccharide between about 1% and about 2% by weight. Particularly preferred seed growth media include between about 0.05% and about 5% by weight of a nitrogen source, such as peptone, between about 0.25% and about 2.5% by weight of autolysed yeast or yeast extract, about 0.5% by weight and about 5% by weight. Glucose between% and phosphorus sources between about 0.05% and about 0.5% by weight, such as monobasic ammonium phosphate. Particularly economical operation of the method is between about 0.5% by weight and about 5% by weight of cornstalk liquid, between about 0.25% by weight and about 2.5% by weight of self-dissolved yeast or yeast extract, about 0.5% by weight and about 5% by weight Obtained by using glucose between and other preferred seed cultures containing between about 0.05% and about 0.5% by weight monobasic ammonium phosphate. Constipates are particularly economical sources of proteins, peptides, carbohydrates, organic acids, vitamins, metal ions, traces and phosphates. Instead of or in addition to the corn steep liquor, mash liquors obtained from other grains may be used. The pH of the medium is preferably adjusted within the range between about 5.0 and about 7.0, for example by addition of alkali metal hydroxides or orthophosphoric acid. If the corn steep liquor is used as a source of nitrogen and carbon, the pH is preferably adjusted between about 6.2 and about 6.8. The medium containing peptone and glucose is preferably adjusted to a pH between about 5.4 and about 6.2. Among the growth media useful for seed fermentation are:

1. Medium # 1: 2% peptone, 2% autolysed yeast (or yeast extract), and 2% glucose; pH was adjusted to 5.8 with 20% NaOH.

2. Medium # 2: 3% corn steep solution, 1.5% yeast extract, 0.3% monobasic ammonium phosphate, and 3% glucose; pH was adjusted to 6.5 with 20% NaOH.

Spores of microorganisms are typically introduced into the medium from vials containing as much as 10 ⁹ spores per ml of suspension. Optimal productivity of seed generation is achieved when dilution with growth medium at the beginning of seed culture does not reduce the density of the spore population to less than about 10 ⁷ per ml. Preferably, the spores are incubated in the seed fermentation system until the packed mycelium volume (PMV) in the seed fermenter is at least about 20%, preferably about 35% to about 45%. Since the cycle in the seed fermentation vessel (or any of the vessels containing the seed fermentation train) depends on the initial concentration of the vessel, providing two or three vessel fermentation steps to speed up the overall process. It may be desirable. However, it is desirable to avoid using three or more more seed fermenters in series, as seed fermentation may impair activity if performed through too many steps. Seed culture fermentation is carried out under stirring at a temperature in the range of about 23 ° C. to about 37 ° C., preferably in the range of about 24 ° C. to about 28 ° C.

Cultures from the seed fermentation system are introduced into the production fermentor along with the production growth medium. In one embodiment of the invention, the non-sterile canrenones or other substrates of formula XIII serve as substrates for the reaction. Preferably, the substrate is added to the resulting fermentor in the form of a slurry of 10% to 30% by weight in the growth medium. In order to increase the surface area that can be utilized in the 11α-hydroxylation reaction, the particle size of the substrate of formula XIII is reduced by passing through an offline differentiator before the substrate is introduced into the fermentor. Sterile nutrient supply stocks containing glucose, and second sterile nutrient solutions (or equivalent amino acid formulations based on other sources such as solubles in distillers) containing yeast derivatives such as self-dissolved yeast are also separately Is introduced. The medium may contain between about 0.5% by weight and about 5% by weight glucose, between about 0.05% and about 5% by weight nitrogen sources, such as peptone, between about 0.05% and about 0.5% by weight phosphorus sources, such as ammonium or alkali Metal phosphates, such as dipotassium hydrogen phosphate, between about 0.25 wt% and about 2.5 wt% yeast lysate or extract (or other amino acid source, such as, for example, a soluble material of a still), about 1 wt% and about 2 wt% Aqueous mixtures containing agar or other non-digestible polysaccharides between% are included. Particularly preferred product growth media include between about 0.05% and about 5% by weight nitrogen source, such as peptone, between about 0.25% and about 2.5% by weight of autolysed yeast or yeast extract, about 0.5% by weight and about 5% by weight. Glucose between% and phosphorus sources between about 0.05% and about 0.5% by weight, such as monobasic ammonium phosphate. Other preferred production media include between about 0.5% by weight and about 5% by weight of cornstalk liquid, between about 0.25% by weight and about 2.5% by weight of autolysed yeast or yeast extract, between about 0.5% by weight and about 5% by weight Glucose and between about 0.05 weight percent and about 0.5 weight percent monobasic ammonium phosphate. The pH of the resulting fermentation medium is preferably adjusted in the manner described above for the seed fermentation medium, to the same preferred pH ranges for the peptone / glucose basal medium and the cornsip liquor basal medium, respectively. Useful growth media for biotransformation include:

1. Medium # 1: 2% peptone, 2% autolysed yeast (or yeast extract), and 2% glucose; pH was adjusted to 5.8 with 20% NaOH.

2. Medium # 2: 1% peptone, 1% autolysed yeast (or yeast extract), and 2% glucose; pH was adjusted to 5.8 with 20% NaOH.

3. Medium # 3: 0.5% peptone, 0.5% autolysed yeast (or yeast extract), and 0.5% glucose; pH was adjusted to 5.8 with 20% NaOH.

4.Medium # 4: 3% corn steep solution, 1.5% yeast extract, 0.3% monobasic ammonium phosphate, and 3% glucose; pH was adjusted to 6.5 with 20% NaOH.

5. Medium # 5: 2.55% cornsip solution, 1.275% yeast extract, 0.255% monobasic ammonium phosphate, and 3% glucose; pH was adjusted to 6.5 with 20% NaOH.

6. Medium # 6: 2.1% corn steep solution, 1.05% yeast extract, 0.21% monobasic ammonium phosphate, and 3% glucose; pH was adjusted to 6.5 with 20% NaOH.

The non-sterile canrenone and sterile nutrient solutions are fed chainwise to the production fermentation in about 5 parts to about 20 parts, preferably about 10 parts to about 15 parts, preferably substantially equal parts, respectively, for the production batch cycle. do. Advantageously, the substrate is initially introduced before inoculation into the seed fermentation broth in an amount sufficient to achieve a concentration between about 0.1% and about 3% by weight, preferably between about 0.5% and about 2% by weight, Then periodically, it is conveniently added at a cumulative ratio between about 1% and about 8% by weight every 8 to 24 hours. If additional substrates are added every 8 hours, the total addition will be slightly lower, such as 0.25 to 2.5 weight percent, as compared to when the substrate is added only every day. In the latter case, cumulative canrenone addition needs to be made in the range of 2% by weight to about 2% by weight. The supplemental nutrient mixture supplied during the fermentation reaction is concentrated, for example between about 40% and about 60% by weight sterile glucose, and between about 16% and about 32% by weight sterile yeast extract or yeast derivative Mixtures containing sterile sources (or other amino acid sources). Since the substrate fed to the production fermenter of FIG. 1 is not sterile, antibiotics are periodically added to the fermentation broth to control the growth of unwanted organisms. Antibiotics such as kanamycin, tetracycline, and kepalexin can be added without adversely affecting growth and in vivo conversion. Preferably, these antibiotics are introduced into the fermentation broth at a concentration between about 0.0004% and about 0.002% based on the total amount of juice, of which between about 0.0002% and about 0.0006 kanamycin sulfate is based on the total amount of juice. At a concentration between%, tetracycline HCl is included at a concentration between about 0.0002% and about 0.006%, and kefalexin is included at a concentration between about 0.001% and about 0.003%.

Typically, the resulting fermentation batch cycle is on the order of about 80 to 160 hours. Therefore, each portion of the formula XIII substrate and nutrient solution is typically added every 2 to 10 hours, preferably every 4 to 6 hours. Advantageously, antifoaming agents are also incorporated into the seed fermentation system and into the resulting fermentor.

In the method of FIG. 1, it is preferred that the inoculum filling the resulting fermentor is from about 0.5 to about 7 volume%, more preferably from about 1 to about 2 volume%, based on the total mixture in the fermentor, glucose The concentration is preferably maintained between about 0.01 and about 1.0 weight%, preferably between about 0.025 and about 0.5 weight%, more preferably between about 0.05 and about 0.25 weight%, wherein glucose is about 0.05 based on the total batch fill It is preferably added periodically at a rate of from about 0.25% by weight. The fermentation temperature is conveniently adjusted in the range of about 20 ° C. to about 37 ° C., preferably about 24 ° C. to about 28 ° C., but the packed mycelium volume (PMV) is below about 60%, more preferably about 50%. In order to maintain below%, it may be desirable to increase the temperature by 2 ° C during the reaction, thereby preventing the viscosity of the fermentation broth from interfering with satisfactory mixing. If biomaterial growth extends above the liquid surface, the substrate retained in the biomaterial can be transported out of the reaction bath, making it unavailable for hydroxylation reactions. For productivity, PMV preferably reaches a range of 30-50%, preferably 35-45% within the first 24 hours of the fermentation reaction, after which to control further growth within the above mentioned limits. It is desirable that the conditions be managed. During the reaction, the pH of the fermentation medium is adjusted between about 5.0 and about 6.5, preferably between about 5.2 and about 5.8, and the fermentor is stirred at a speed of about 400 rpm to about 800 rpm. The dissolved oxygen level is at least about 10% of saturation by venting the batch between about 0.2 and about 1.0 vvm, and the pressure in the top space of the fermentor is from atmospheric to about 1.0 bar gauge, most preferably around 0.7 bar gauge. Is maintained at. Agitation rates may also be increased as needed to maintain minimal dissolved oxygen levels. Beneficially, dissolved oxygen remains at least about 10%, in fact as high as about 50%, to facilitate the conversion of the substrate. Maintaining the pH at 5.5 ± 0.2 is also most suitable for bioconversion. Foaming is adjusted as needed by the addition of conventional antifoaming agents. After all the substrates have been added, the reaction preferably continues until the molar ratio of the formula XIII product to the remaining unreacted formula XIII substrate is at least 9 to 1. Such bioconversion may be accomplished in an 80 to 160 hour batch cycle as mentioned above.

Higher levels of conversion are associated with depletion of the initial nutrient levels below the initial fill level and are known to prevent the substrate from popping out of the liquid broth by controlling the rate of aeration and stirring. In the method of FIG. 1, the nutrient level is kept up to about 60% or less, preferably about 50% or less of the initial fill level; In the method of FIGS. 2 and 3, the nutrient level was reduced to remain at about 80% or less, preferably about 70% or less of the initial fill level. The ventilation rate is preferably in the range of 1 vvm or less, more preferably about 0.5 vvm; It is preferable that the stirring speed is 600 rpm or less.

Particularly preferred methods for the preparation of formula VIII are illustrated in FIG. 2. A preferred microorganism for 11α-hydroxylation of a compound of formula XIII (such as canrenone) is Aspergillus okraceus NRRL 405 (ATCC 18500). In this method, the growth medium preferably contains between about 0.5% and about 5% by weight of cornstalk liquid, between about 0.5% by weight and about 5% by weight of glucose, between about 0.1% by weight and about 3% by weight of yeast extract. And between about 0.05% and about 0.5% by weight of ammonium phosphate. However, other production growth media as described herein may also be used. Seed cultures are prepared essentially using any of the seed fermentation media described herein, in the manner described for the method of FIG. 1. Suspensions of non-micronized canrenone or other substrates of formula XIII in the growth medium are prepared aseptically in a mixer, preferably at relatively high concentrations of about 10% and about 30% by weight of the substrate. Preferably, the aseptic preparation includes sterilization or pasteurization of the suspension after mixing. The total amount of sterile substrate suspension required for the production batch is introduced into the production fermentor at the beginning of the batch or by periodic chain feeding. The particle size of the substrate is reduced by wet milling in an on-line shear pump that delivers the slurry to the production fermentor, thereby eliminating the need for using an off-line micronizer. Where aseptic conditions are achieved by pasteurization rather than by sterilization, the degree of aggregation may be insignificant, but the use of a shear pump may be desirable to provide a positive control of particle size. Sterile growth medium and glucose solution are introduced into the resulting fermentor in essentially the same manner as described above. All components supplied to the production fermenter are sterilized prior to introduction, thereby eliminating the need for antibiotics.

Preferably, during operation of the method of FIG. 2, the inoculum is preferably introduced into the product fermentor at a rate between about 0.5% to about 7%, and the fermentation temperature is about 20 ° C to about 37 ° C, preferably about 24 Preferably between about 4.5 and about 6.5, preferably between about 5.3 and about 5.5, such as by gas ammonia, aqueous ammonium hydroxide, aqueous alkali metal hydroxide, or orthophosphoric acid. As in the method of FIG. 1, the temperature is preferably adjusted to control the growth of the biomaterial so that the PMV does not exceed 55-60%. The initial glucose filling is preferably between about 1% by weight and about 4% by weight, most preferably between about 2.5% by weight and about 3.5% by weight, but is preferably left to fall below about 1.0% by weight during fermentation. . Supplementary glucose is periodically supplied at a rate between about 0.2% and about 1.0% by weight based on the total batch fill, such that the glucose concentration in the fermented coarse eye is between about 0.1% and about 1.5% by weight, preferably about 0.25% by weight. To about 0.5% by weight. Optionally, nitrogen and phosphorus sources can be supplemented with glucose. However, since the overall canrenone filling is made at the beginning of the batch cycle, the necessary supplements of nitrogen and phosphorus containing nutrients can also be introduced at that time, so that only glucose solution can be used for supplementation during the reaction. The speed and nature of the agitation is quite variable. Moderately vigorous stirring promotes the transfer of material between the solid substrate and the aqueous phase. However, in order to prevent microorganisms from degrading myelin, a low shear force impeller must be used. The optimum stirring speed is affected by culture broth viscosity, oxygen concentration, and container, baffle and impeller morphology, and varies within the range of 200 to 800 rpm depending on the mixing conditions. Typically, the preferred stirring speed is in the range of 350 to 600 rpm. Preferably the stirring impeller provides a function of pumping along the axis by shaping the lower side to aid good mixing of the fermented biomaterials. The batch is preferably vented at a speed of about 0.3 to about 1.0 vvm, preferably about 0.4 to about 0.8 vvm, and the pressure in the top space of the fermentor is preferably about 0.5 to about 1.0 bar gauge. The temperature, stirring, ventilation and back pressure are preferably adjusted to maintain dissolved oxygen in the range of at least about 10% by volume during in vivo conversion. The total batch cycle is typically about 100 to about 140 hours.

Although the principle of operating the method of FIG. 2 is substantially based on the initial introduction of overall canlenone filling, it will be appreciated that the growth of fermentation gravy can be performed before the bulk canrenone is filled. Optionally, part of canrenone may also be added later to the batch. Generally, however, at least about 75% of the sterilized canlenone filling should be introduced into the conversion fermentor within 48 hours after the start of the fermentation. Moreover, at least about 25% by weight of canrenone is preferably introduced at the start of the fermentation, or at least within the first 24 hours, to facilitate production of the bioconversion enzyme (s).

In a further preferred method as illustrated in FIG. 3, the entire batch fill and nutrient solution are sterilized in the resulting fermentation vessel before the inoculum is introduced. Nutrient solutions that can be used will be preferred among them, but are essentially the same as in the method of FIG. 2. In this embodiment of the invention, the shearing action of the stirrer impeller destroys substrate aggregates which otherwise tend to form upon sterilization. If the average particle size of canrenone is less than about 300 μ and at least 75% by weight of the particles is less than 240 μ, the reaction proceeds satisfactorily. The use of a suitable impeller, for example a disk turbine impeller, at a suitable speed in the range of 200 to 800 rpm, at a tip speed of at least about 400 cm / sec, despite the agglomeration which tends to occur when sterilized in the resulting fermenter And to provide sufficient shear rate to maintain such particle size properties. The remaining operation of the method of FIG. 3 is essentially the same as the method of FIG. The methods of FIGS. 2 and 3 provide several distinct advantages over the method of FIG. 1. A particular advantage is the possibility of using low-cost nutrient bases such as cornstalk liquids. Further elimination of the need for antibiotics further simplifies the feeding process and realizes the additional advantage of enabling batch sterilization of canrenone or other substrates of formula XIII. Another advantage is the ability to use simple glucose solutions rather than complex nutrient solutions for supplementation during the reaction cycle.

In the methods shown in FIGS. 1-3, the product of formula VIII is a crystalline solid that can be separated with the biomaterial from the reaction broth by filtration or low speed centrifugation. Alternatively, the product can be extracted from the whole reaction broth using an organic solvent. The product of formula VIII is recovered by solvent extraction. For maximum recovery, both the liquid phase filtrate and the biomaterial filter or centrifugal cake are both treated with extraction solvent, but usually at least 95% of the product is bound to the biomaterial. Typically, hydrocarbons, esters, chlorinated hydrocarbons, and ketone solvents can be used for extraction. Preferred solvent is ethyl acetate. Other typical suitable solvents are toluene and methyl isobutyl ketone. In order to extract from the liquid phase, it is convenient to use a solvent to which the reaction solution of a volume approximately equal to the volume of the reaction solution comes into contact. In order to recover the product from the biomaterial, the product is suspended in a solvent, preferably in a volume of 50-100 ml of solvent per gram of the initial canrenone charge, relative to the initial charge of the substrate, and the resulting suspension is preferred. Preferably, the product is refluxed for about 20 minutes to several hours to ensure that the product is transported from the deeper portions and holes of the biomaterial onto the solvent. The biomaterial is then removed by filtration or centrifugation, and the filter cake is preferably washed with both fresh solvent and deionized water. The aqueous and solvent washes are then left to separate and combined. The product of formula VIII is recovered from solution by crystallization. To maximize the yield, the mycelium is contacted twice with fresh solvent. After allowing the separation of the aqueous phase to settle, the product is recovered from the solvent phase. More preferably, the solvent is removed under vacuum until crystallization begins, and then the concentrated extract is brought to a temperature of 0 to about 20 ° C., preferably at a temperature of about 10 to about 15 ° C., for a time sufficient for crystal precipitation and growth. Typically for about 8 to about 12 hours.

Particularly preferred is the method of FIG. 2 and in particular the method of FIG. 3. These methods operate at low viscosities and are much easier to control the parameters of the method such as pH, temperature and dissolved oxygen. Moreover, sterile conditions can be readily preserved without frequent use of antibiotics.

The bioconversion method is exothermic so that heat must be removed using a cooling coil in a jacketed fermenter or a product fermenter. Alternatively, the reaction broth can be circulated through an external heat exchanger. Dissolved oxygen is preferably at a level of at least about 5% by volume, preferably at least about 10% by volume, sufficient to provide energy to the reaction and to ensure the conversion of glucose to CO ₂ and H ₂ O This is maintained by controlling the rate of air introduced into the reactor in response to the measurement of the potential. The pH is preferably adjusted between about 4.5 and about 6.5.

In each of the alternative methods for 11-hydroxylation of the substrate of formula XIII, productivity is limited by mass transfer from the solid substrate to the aqueous phase, or the interface at which the reaction is believed to occur. As indicated above, productivity is not significantly limited by the mass transfer rate as long as the average particle size of the substrate particles is reduced to at least about 300 μ or less and at least 75% by weight of the particles are smaller than 240 μ. However, the productivity of these methods can be further enhanced in certain alternative embodiments in which a substantial charge of canrenone or other substrate of formula XIII is provided in the production fermentor in an organic solvent. In one option, the substrate is dissolved in a solvent that is incompatible with water and mixed with an aqueous growth medium inoculum and surfactant. Solvents that are incompatible with water include, for example, DMF, DMSO, C ₆ -C ₁₂ fatty acids, C ₆ -C ₁₂ n-alkanes, vegetable oils, sorbitan, and aqueous surfactant solutions. Stirring of this charge results in an emulsion reaction system with increased interfacial area for mass transfer of the substrate from the organic liquid phase to the reaction site.

The second option is to dissolve the substrate in a solvent mixed with water, such as acetone, methylethyl ketone, methanol, ethanol, or glaserol, at a concentration substantially greater than its solubility in water. By preparing the initial substrate solution at elevated temperature, the solubility is increased, thereby further increasing the amount of substrate in solution form introduced into the reactor and ultimately enhancing the reactor payload. Warm substrate solution is charged to the resulting fermentation reactor with a relatively low temperature aqueous charge containing growth medium inoculum. When the substrate solution is mixed with the aqueous medium, precipitation of the substrate occurs. However, under substantial supersaturation conditions and moderately vigorous stirring, nucleation takes precedence over crystal growth, and very fine particles with a large surface area are formed. The large surface area promotes mass transfer between the liquid phase and the solid substrate. Moreover, the equilibrium concentration of the substrate in the aqueous liquid phase is also enhanced in the presence of a solvent that can be mixed with water. Thus, productivity is promoted.

Although microorganisms are not necessarily able to tolerate high concentrations of organic solvents in the aqueous phase, ethanol concentrations, for example in the range of about 3% to about 5% by weight, can be advantageously used.

The third option is to dissolve the substrate in aqueous cyclodextrin. Exemplary cyclodextrins include hydroxypropyl-β-cyclodextrin and methyl-β-cyclodextrin. The molar ratio of substrate: cyclodextrin is preferably from about 1: 0.5 to about 1: 1.5, more preferably from about 1: 0.8 to about 1: 1. The substrate: cyclodextrin mixture can then be added to the bioconversion reactor aseptically.

11α-hydroxykanrenone and other products of the 11α-hydroxylation method (Formulas VIII and VIIIA) are novel compounds that can be isolated by filtering the reaction medium and extracting the product from the biomaterial collected on the filter medium. Conventional organic solvents such as ethyl acetate, acetone, toluene, chlorinated hydrocarbons, and methyl isobutyl ketone can be used for extraction. The product of formula VIII can then be recrystallized from organic solvents of the same type. Compounds of formula VIII have substantial value as intermediates for the preparation of compounds of formula I, in particular compounds of formula IA.

Preferably, the compound of formula VIII is wherein -AA- and -BB- are -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20-spiroxane Corresponds to formula VIIIA, which forms a ring:

(Formula XXXIII)

Also according to the method of method 1, the compound of formula VIII is reacted with a source of cyanide ions under alkaline conditions to give an enamine compound of formula VII:

Formula VII

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined above.

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

Wherein, -AA-, -BB-, R ³ , Y ¹ , Y ² , and X are as defined for formula XIIIA above. R ³ is preferably hydrogen.

Cyanation of the 11α-hydroxyl substrate of formula VIII is carried out by reacting it in the presence of a base and an alkali metal salt, most preferably LiCl, with a cyanide ion such as ketone cyanohydrin, most preferably acetone cyanohydrin Can be. Alternatively, cyanation can be accomplished by using alkali metal cyanide in the presence of an acid without cyanohydrin.

In the ketone cyanohydrin process, the reaction is carried out in solution, preferably with an aprotic polar solvent such as dimethylformamide or dimethyl sulfoxide. Formation of enamines requires at least 2 moles of cyanide ion source per mole of substrate, preferably with slightly more cyanide sources. The base is preferably a nitrogen containing base such as dialkylamine, trialkylamine, alkanolamine, pyridine and the like. However, inorganic bases such as alkali metal carbonates or alkali metal hydroxides can also be used. Preferably, the substrate of formula VIII is initially present at a ratio between about 20% and about 50% by weight and the base is present at a rate of 0.5 to 2 equivalents per equivalent of substrate. The reaction temperature is not critical but productivity is enhanced by operating at elevated temperatures. Thus, for example, when triethylamine is used as the base, the reaction is advantageously carried out in the range of about 80 ° C to about 90 ° C. At such temperatures, the reaction completes in about 5 to about 20 hours. If diisopropylethyl is used as the base and the reaction is carried out at 105 ° C., the reaction is terminated at 8 hours. At the end of the reaction period, the solvent is removed in vacuo and neutralized to pH 7 with diluted acid, preferably hydrochloric acid. From this solution the product precipitates, which is then washed with distilled water and air dried. The released HCN can be stripped with inert gas and suppressed in alkaline solution. The dried precipitate is taken up in chloroform or other suitable solvent and then extracted with concentrated acid such as 6 N HCl. The extract is neutralized to pH 7 by addition of an inorganic base, preferably alkali metal hydroxide, and cooled to a temperature of 0 ° C. The resulting precipitate is washed and dried and then recrystallized from a suitable solvent such as acetone to give a product of formula VII suitable for use in the next step of the process.

Alternatively, the reaction can be carried out in an aqueous solvent system comprising an organic solvent, such as methanol, which can be mixed with water, or in a two-phase system comprising water and an organic solvent, such as ethyl acetate. In this alternative method, the product is diluted with water and then the product is extracted using an organic solvent such as methylene chloride or chloroform and then extracted again from the organic extract using a concentrated mineral acid such as 2N HCl. [See US Pat. No. 3,200,113].

According to another alternative method, the reaction can be carried out in a solvent which can be mixed with water such as dimethylformamide, dimethylacetamide, N-methyl, pyrrolidone or dimethyl sulfoxide, and the reaction product solution is then watered. Dilution is made alkaline, for example by addition of alkali metal carbonate, and then cooled to 0 ° C to 10 ° C to cause precipitation of the product. Preferably, the system is inhibited with alkali metal hypohalite or other agent effective to inhibit the occurrence of cyanide. After filtration and washing with water, the precipitated product is suitable for use in the next step of the process.

According to another alternative method, the enamine product of formula VII can react the substrate of formula VIII in the presence of a proton source with an excess of alkali metal cyanide, preferably NaCN and aprotic water- such as dimethylformamide or dimethylacetamide. It can be produced by reacting in an aqueous solvent containing a miscible polar solvent. The proton source is preferably mineral or C ₁ to C ₅ carboxylic acid, sulfuric acid being particularly preferred. In exceptional cases where the cyanide reagent is LiCN in commercial DMF, no separate proton source needs to be added.

The source of cyanide ions, such as alkali metal salts, is preferably charged to the reactor at a ratio of about 2.05 to about 5 molar equivalents per equivalent of substrate. The mine or other proton source is believed to promote the addition of HCN across the double bonds of 4,5 and 6,7 and is preferably present at a rate of at least 1 molar equivalent per molar equivalent of the substrate; However, the reaction system must remain basic by keeping excess alkali metal cyanide in excess of the acid present. The reaction is preferably carried out at a temperature of at least about 75 ° C., typically 60 ° C. to 100 ° C., for about 1 to 8 hours, preferably for about 1.5 to about 3 hours. At the end of the reaction period, the reaction mixture is preferably cooled to approximately room temperature, and the product enamine precipitates by acidifying the reaction mixture and mixing it with cold water, preferably at temperatures around the ice bath temperature. Acidification is believed to close 17-lactone, which tends to open under basic conditions predominant in cyanation. The reaction mixture is conveniently acidified with the same acid present in the reaction, preferably sulfuric acid. Water is preferably added at a rate of about 10 to about 50 molar equivalents per mole of product.

The compounds of formula VII are novel compounds and have substantial value as intermediates for the preparation of compounds of formula I, in particular compounds of formula IA. Preferably, the compound of formula VII is -AA- and -BB- is -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy and R ⁸ and R ⁹ together are 20-spir Corresponds to the formula VIIA constituting the roxane ring:

(Formula XXXIII)

Most preferred compounds of formula VII are 5'R (5'α), 7'β-20'-aminohexadecahydro-11'β-hydroxy-10'α, 13'α-dimethyl-3 ', 5- Dioxopyro [furan-2 (3H), 17'α (5'H)-[7,4] metheno [4H] cyclopenta [a] phenanthrene] -5'-carbonitrile.

In the conversion of the compound of formula VIII to the enamine of formula VII, a 7-cyano derivative of the compound of formula VIII was observed by chromatography in the crude product. 7-cyano compounds are believed to be intermediates in the conversion process. It was also assumed that the 7-cyano intermediate itself reacts to form a second intermediate which is the 5,7-cyano derivative of the compound of formula VIII, and subsequently reacts to form the esters [R. Christiansen et al., The Reaction of Steroidal 4,6-Dien-3-Ones With Cyanide, Steroids , Vol. 1, June 1963]. These new compounds are also utilized as markers on chromatography as well as synthetic intermediates. Total Method 1 In a preferred embodiment at this stage of the synthetic method, these intermediates are 7α-cyano-11α, 17-dihydroxy-3-oxo-17α-pregin-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 step of the synthesis of Method 1, the enamine of formula VII is hydrolyzed to yield the diketone compound of formula VI:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII. Any aqueous organic acid or photoacid can be used for hydrolysis. Hydrochloric acid is preferred. In order to enhance the productivity, it is preferable to use an organic solvent mixed with water such as dimethylacetamide or lower alkanol as a solvent. More preferred solvent is dimethylacetamide. The acid should be present in a proportion of at least one equivalent per equivalent of the substrate of formula VII. In aqueous systems, the enamine substrate VII can be substantially converted to the diketone of formula VI at about 80 ° C. for a period of about 5 hours. Operation at elevated temperatures increases productivity, but temperature is not a decisive factor. Suitable temperatures are selected based on the volatility of the solvent system and the acid.

Preferably, the enamine substrate of formula VII corresponds to formula VIIA and the diketone product corresponds to formula VIA:

Formula VIIA

And

In these two formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² and X are as defined in formula XIIIA. R ³ is preferably hydrogen.

At the end of the reaction period, the solution is cooled to 0 to about 25 ° C. to crystallize the product. The product crystals are recrystallized from a suitable solvent such as isopropanol or methanol to give the product of formula VI suitable for use in the next step of the process; However, recrystallization is not necessary. The product of formula VI is of substantial value as an intermediate for preparing compounds of formula I, in particular formula IA. Preferably, the compound of formula VI is -AA- and -BB- is -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20-speech Corresponds to the formula VIA constituting the roxane ring:

(Formula XXXIII)

Most preferred compounds of formula VI are 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-trioxospyro [Furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile.

In a particularly preferred embodiment of the invention, the product enamine of formula VII is produced from the compound of formula VIII in the manner described above and converted in situ to the diketone of formula VI. In this embodiment of the invention, the substrate of formula VIII is reacted with an excess of alkali metal cyanide in an aqueous solvent containing a proton source as described above or optionally with an excess of ketone cyanohi in the presence of a base and LiCl. Reacts with offered However, instead of water being added at a rate calculated to cause precipitation of the enamines after the reaction mixture has cooled and acidified, it is preferred to avoid substantial cooling of the reaction mixture. Instead water and an acid, preferably a mineral acid such as sulfuric acid, are added to the mixture at the end of the cyanation reaction. The proportion of acid added is sufficient to neutralize excess alkali metal cyanide, typically at least 1 molar equivalent per mole of formula VIII substrate, preferably about 2 to 5 molar equivalents per equivalent of substrate . However, the temperature is kept sufficiently high, the dilution rate is sufficiently large to prevent substantially precipitation and it is possible for the hydrolysis of the enamine to diketone to proceed in the liquid phase. Therefore, the method proceeds with minimal interference and high productivity. The hydrolysis is preferably at a temperature in the range of at least 80 ° C., more preferably from about 90 ° C. to about 100 ° C., typically from about 1 hour to about 10 hours, more preferably from about 2 hours to about 5 hours. Is performed for a period of time. The reaction mixture is then preferably cooled to a temperature between 0 ° C. and 15 ° C., advantageously in an ice bath to a temperature of about 5 ° C. to about 10 ° C. to precipitate the product diketone of formula VI. Solid product can be recovered by filtration and impurities can be reduced by washing with water.

As a next step in the synthesis of method 1, the diketone compound of formula VI reacts with the metal alkoxide to open the ketone bridge between positions 4 and 7 through the separation of the bond between the carbonyl group and the 4-carbon to position 7 α-oriented alkoxycarbonyl substituents are formed and cyanide at 5-carbon is removed. The product of this reaction is a hydroxyester compound corresponding to formula V:

Formula V

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII and R ¹ is lower alkoxycarbonyl or hydroxycarbonyl. The metal alkoxide used in the reaction corresponds to the formula R ¹⁰ OM, M is an alkali metal, and R ¹⁰ O- corresponds to an alkoxy substituent of R ¹ . The yield of this reaction is most satisfactory when the metal alkoxide is potassium methoxylated or sodium methoxylated, but other lower alkoxides may also be used. Potassium alkoxides are particularly preferred. Phenoxides, other aryloxides may also be used, and aryl sulfides may also be used. The reaction may conveniently be carried out in the presence of an alcohol corresponding to the formula R ¹⁰ OH (wherein R ¹⁰ is as defined above). Other conventional solvents may also be used. Preferably, the substrate of formula VI is present in a ratio between about 2% and about 12% by weight, more preferably at least about 6% by weight. Preferably, R ¹⁰ OM is present at a ratio between about 0.5 to about 4 moles of the substrate, more preferably between about 1 mole and about 2 moles of the substrate, most preferably at a rate of about 1.6 moles of the substrate. Temperature is not critical, but elevated temperatures increase productivity. The reaction time is typically about 4 hours to 24 hours, preferably about 4 hours to 16 hours. Conveniently, the reaction is carried out at reflux temperature of the atmosphere, depending on the type of solvent used.

The time required for the reaction to reach equilibrium is influenced by the amount of alkoxide added to the reaction mixture and the manner in which the alkoxide is added. Alkoxides may be added at one time or in portions, or continuously. If the alkoxide is added in several portions, it is preferred that about 1.6 equivalents of potassium methoxide are added in two steps. In this two-step addition, one equivalent of potassium methoxide is added first and after 90 minutes 0.6 equivalents of potassium methoxide are added. This two-step addition shortens the time to reach equilibrium relative to adding 1.6 equivalents of potassium methoxide at one time.

Since equilibrium is more favorable for the production of hydroxyesters at lower concentrations of diketones, the reaction is preferably operated at higher dilution rates, such as 40: 1 for reactions with sodium methoxide. Significantly higher productivity can be achieved by using potassium methoxide rather than sodium methoxide because a dilution in the range of about 20: 1 is generally sufficient to minimize the degree of reverse cyanation when potassium methoxide is the reagent.

In accordance with the present invention, it has been found that the reverse cyanation reaction can be inhibited by taking appropriate chemical or physical measures to remove byproduct cyanide ions from the reaction coarseness. Therefore, in another embodiment of the invention, the reaction of the diketone with the alkali metal alkoxide may be carried out in the presence of a salt causing a precipitation inducing agent for cyanide ions, such as a cation forming an insoluble cyanide compound. Such salts include, for example, zinc iodide, iron sulfate, or salts of transition metals that are essentially more soluble than all halides, sulfates or other alkaline earth metals or corresponding cyanides. If zinc iodide is present at a rate of about 1 equivalent per equivalent of diketone substrate, the productivity of the reaction has been observed to be substantially increased compared to the method performed in the absence of alkali metal halides.

Even when precipitation inducers are used for the removal of cyanide ions, it is still desirable to carry out the method at a fairly high dilution rate, but the molar ratio of solvent to diketone substrate by using precipitation incidence reactions in the absence of such agents Will be significantly reduced in comparison with. Recovery of the hydroxyesters of formula V can be carried out according to any of the extractive or non-extracting procedures described below.

The equilibrium of the reaction can also be adjusted to favor the production of the hydroxyester of formula V by removing it from the reaction mixture after the hydroxyester has been synthesized. Removal of the hydroxyester may proceed stepwise or continuously through means such as filtration. Removal of hydroxyesters can be used alone or in combination with chemicals or to control the equilibrium with physical removal of cyanide from the reaction mixture. Heating the resulting filtrate then causes the reaction equilibrium to preferentially convert the remaining diketone of formula VI to the hydroxyester of formula V more preferentially.

In the conversion of the diketone of formula VI to the hydroxyester of formula V, it was observed that 5-cyano hydroxyester is present in the crude product in small amounts, typically up to about 5% by weight. 5-Cyano hydroxyester is believed to be an equilibrium intermediate between the diketone of formula VI and the hydroxyester of formula V. This equilibrium intermediate was also added to the 3-keto- ^Δ4,5 function of the hydroxyester of the hydroxyester of the cyanide ion and by-product cyanide ion through methoxide attack on the 5,7-oxo group and protonation It is believed to form from hydroxyesters through.

In addition, 5-cyano-7-acid and 17-alkoxide of the hydroxyester of formula V were observed in the chromatography of the crude product. It is assumed that the 5-cyano hydroxyester intermediate reacts with the byproduct cyanide ion (which exists as a result of the decyanation to introduce the Δ ^4,5 double bond) to produce 5-cyano-7-acid. The action of cyanide ions is assumed to result in the 5-cyano-7-acid and the corresponding alkylnitrile produced by dealkylation of the 7-ester group of 5-cyano hydroxyester.

It is also believed that transient intermediates of 17-alkoxides are formed from the attack of methoxides on hydroxyesters to 17-spirolactone (or the preceding intermediates continue to be converted to hydroxyesters). 17-alkoxides are readily converted to hydroxyesters when treated with an acid. Therefore, it is not generally observed in the product matrix.

5-cyano hydroxyesters, 5-cyano-7-acids, and 17-alkoxy are novel compounds useful as markers and intermediates on chromatography in the preparation of hydroxyesters. They can be isolated from the crude product at this stage of the synthesis of Method 1. Alternatively, they can be synthesized directly for use as markers or as intermediates. 5-cyano hydroxyesters can be synthesized by reacting a solution of the isolated diketone of formula VI with a base such as an alkoxide or an amine and isolating the resulting precipitate. Preferred compounds are 7-methyl hydrogen 5β-cyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone.

5-Cyano-7-carboxylic acid can be synthesized directly by reacting the diketone of formula VI with a weak aqueous base such as sodium acetate or sodium bicarbonate and then isolating the resulting precipitate. Preferred compounds are 5-β-cyano-11-α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone.

17-alkoxide can be synthesized directly by reacting the hydroxyester solution of formula V with an alkoxide to produce a mixture of 17-alkoxide and the corresponding hydroxyester. Preferred compounds are dimethyl 11α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone.

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

Formula VIA

The hydroxyester product corresponds to the formula VA:

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² and X are as defined for formula XIIIA and R ¹ is as defined in formula V. It is preferable that R ³ is hydrogen.

The product of formula V is a novel compound that has substantial value as an intermediate for the preparation of compounds of formula I, in particular compounds of formula IA. Preferably, the compound of formula V is -AA- and -BB- is -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20-speech of the formula Corresponds to the formula VA constituting the roxane ring:

(Formula XXXIII)

Most preferred compounds of formula V are methyl hydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

Compounds of formula V can be isolated by filtration or by acidifying the reaction solution with, for example, aqueous HCl or sulfuric acid, cooling to ambient temperature, and then extracting the product with an organic solvent such as methylene chloride or ethyl acetate. have. The extract is washed with an aqueous alkaline wash solution, dried and filtered and then the solvent is removed. Alternatively, the reaction solution containing the product of formula V can be inhibited with concentrated acid. The product solution is concentrated and after cooling to 0 ° C. to about 25 ° C., the product solids are isolated by filtration.

In a preferred embodiment, methanol and HCN are removed by distillation with a mineral acid (such as hydrochloric acid or sulfuric acid) added before distillation and water added after distillation after the reaction period is over. The mine can be added in a single step, in multiple steps or continuously. In a preferred embodiment, the mine is added continuously over a period of about 10 to about 40 minutes, more preferably about 15 to about 30 minutes. Likewise, water can be added to the bottom of the distiller in a single stage, in multiple stages or continuously. In a preferred embodiment, the concentrated reaction mixture is cooled from reflux temperature before water is added. Preferably, the mixture is cooled to about 50 to about 70 ° C., more preferably about 60 to about 70 ° C., and most preferably about 65 ° C. before water is added. Water is then added continuously, preferably over a period of about 15 minutes to about 3 hours, more preferably about 60 minutes to about 90 minutes, during which the temperature remains almost constant. The product of formula V begins to crystallize from the bottom of the distiller as water is added. After the water has been added to the mixture, the diluted reaction mixture is maintained at about the same temperature for about 1 hour and then cooled to about 15 ° C. over about 4 to 5 hours. The mixture is maintained at about 15 ° C. for about 1 to 2 hours. Leaving at 15 ° C. for a longer time increases the yield of cyanoester in the mixture. This recovery method provides a high quality crystalline product without extraction operation.

According to another preferred manner of recovering the product of formula V, methanol and HCN are removed by distillation using water and acid added before or during the distillation period. Adding water prior to distillation simplifies operation, but incremental addition prior to distillation allows the volume in the still to remain substantially constant. The product of formula V crystallizes from the bottom of the distiller as distillation proceeds. This recovery method provides a high quality crystal product without extraction operation.

According to a further alternative method, the reaction solution containing the product of formula V can be inhibited with mineral acid, for example 4N HCl, and then the solvent is removed by distillation. Removal of the solvent is also effective to remove residual HCN from the reaction product. When the compound of formula V is used as an intermediate in the process for the preparation of epoxymexrenone as described herein, it has been found that it is not necessary to extract the solvent several times for the purification of the compound of formula V. In practice, such extraction may sometimes not be performed completely. If solvent extraction is used for product purification, it is preferable to supplement the solvent washes with saline and caustic washes. However, if solvent extraction is not carried out, saline and caustic washing are also not necessary. The elimination of the extraction and washing process can significantly increase the productivity of the process without affecting yield or product quality, and also eliminates the need to dry the washed solution with a desiccant such as sodium sulfate.

The crude 11α-hydroxy-7α-alkoxycarbonyl product is placed in a solvent for the next reaction step of the process, ie, the conversion of the 11-hydroxy group to the leaving group at the 11 position results in the compound of formula IV:

Formula IV

Wherein -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula XIII, R ¹ is as defined in formula V, and R ² is lower arylsulfonyloxy, alkyl Sulfonyloxy, acyloxy or halides. Preferred is that 11α-hydroxy is esterified by reaction with lower alkylsulfonyl halides, acyl halides or acid anhydrides added to the solution containing the intermediate product of formula V. Lower acid anhydrides such as acetic anhydride and trihalogenated acid anhydrides such as trifluoroacetic anhydride can be used to prepare suitable acyloxy leaving groups. However, lower alkylsulfonyl halides, in particular methanesulfonyl chloride, are preferred. Alternatively, the 11-α hydroxy group can be converted to a halide by reaction of a suitable reagent such as thionyl bromide, thionyl chloride, sulfuryl chloride or oxalyl chloride. Other reagents for forming the 11α-sulfonic acid esters are tosyl chloride, benzenesulfonyl chloride and trifluoromethanesulfonic anhydride. The reaction is carried out in a solvent containing a hydrogen halide scavenger such as triethylamine or pyridine. Inorganic bases such as potassium carbonate or sodium carbonate can also be used. The initial concentration of the hydroxyester of formula V is preferably from about 5% by weight to about 50% by weight. The esterification reagent is preferably present in slightly excess amount. Methylene chloride is a particularly suitable solvent for the reaction, but other solvents such as dichloroethane, pyridine, chloroform, methyl ethyl ketone, dimethoxyethane, methyl isobutyl ketone, acetone, other ketones, ethers, acetonitrile, toluene and tetrahydrofuran This can also be used. The reaction temperature is mainly determined by the volatility of the solvent. In methylene chloride, it is preferable that reaction temperature exists in the range of about -10 degreeC to about 10 degreeC.

Preferably, the hydroxyester substrate of formula V corresponds to formula VA:

Formula VA

The product corresponds to the formula IVA:

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² and X are as defined in formula XIIIA, R ¹ is lower alkoxycarbonyl or hydroxycarbonyl, and R ² is formula IV As defined in. R ³ is preferably hydrogen.

The product of formula IV is a novel compound that has substantial value as an intermediate for the preparation of compounds of formula I, in particular compounds of formula IA. Preferably, the compound of formula IVA is -AA- and -BB- is -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20-speech of the formula Corresponds to the formula VA constituting the roxane ring:

(Formula XXXIII)

Most preferred compounds of formula IV are methyl hydrogen 17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone. If an acyloxy leaving group is required, the compound of formula IV is 7-methyl hydrogen 17-hydroxy-3-oxo-11α- (2,2,2-trifluoro-1-oxoethoxy) -17α-pre Near-4-ene-7α, 21-dicarboxylate, γ-lactone; Or 7-methyl 11α- (acetyloxy) -17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone.

If desired, the compound of formula IV can be isolated by removing the solvent. Preferably, the reaction solution is first washed with an aqueous alkaline washing solution, such as 0.5 to 2 N NaOH, followed by an acidic washing solution such as 0.5 to 2 N HCl. After the reaction solvent is removed, the product is recrystallized by, for example, taking the product in methylene chloride and then adding another solvent, such as ethyl ether, which reduces the solubility of the product of formula IV, causing the product of formula IV to precipitate in crystalline form. .

In the recovery of the product of formula IV or in the preparation of a reaction solution for the conversion of an intermediate of formula IV to an intermediate of formula II as further described below, all extraction and / or washing steps can be carried out if the solution is acidic and It may not be done if it is instead treated with an ion exchange resin to remove basic impurities. The solution is first treated with an anion exchange resin and then with a cation exchange resin. Alternatively, the reaction solution may first be treated with an inorganic adsorbent such as basic alumina or basic silica and then with a diluted acid wash. Basic silica or basic alumina may be mixed with the reaction solution, typically at a rate between about 5 and about 50 g per kg of product, preferably at a rate of about 15 to about 20 g per kg of product. If an ion exchange resin or inorganic adsorbent is used, the treatment can be carried out simply by making a slurry with the reaction solution under conditions of stirring the resin or inorganic adsorbent at ambient temperature, and then removing the resin or inorganic adsorbent by filtration.

In another preferred embodiment of the invention, the product compound of formula IV is recovered in crude form as a concentrated solution by removal of the solvent moiety. This concentrated solution is used directly in the next step of the process, in which the ester of formula II is prepared by removing the 11α-leaving group from the compound of formula IV:

Wherein, -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII above and R ¹ is as defined in formula V. For the purpose of this reaction, the R ² substituent of the compound of formula IV can be any leaving group, the separation of which is effective to make a double bond between 9- and 11-carbon. Preferably, the leaving group is a lower alkylsulfonyloxy or acyloxy substituent, which is removed by reaction with an acid and an alkali metal salt. Mines may be used, but lower alkanoic acids are preferred. Advantageously, the reagents for the reaction further comprise alkali metal salts of alkanes which are used. It is particularly preferred that the leaving group comprises mesyloxy and the reaction reagent comprises an alkali metal salt of formic acid or acetic acid and one of these acids or other lower alkanoic acids. When the leaving group is mesyloxy and the removal reagent is acetic acid and sodium acetate or formic acid and potassium formate, a relatively high ratio of 9,11-olefins to 11,12-olefins is observed. If free water is present while the leaving group is being removed, impurities tend to form 7,9-lactones of the following formula, which compounds are difficult to remove from the final product:

Wherein -AA-, R ³ , -BB-, R ⁸ and R ⁹ are as defined in formula XIII. Therefore acetic anhydride or other desiccant is used to remove the water present in the formic acid. The free water content of the reaction mixture before the reaction should be maintained at levels below about 0.5% by weight, preferably below 0.1% by weight, based on the total reaction solution as determined by Karl Fisher analysis of water. . Although it is desirable to keep the reaction mixture dry enough to carry out the reaction, satisfactory results have been achieved with 0.3% by weight of water. Preferably, the reaction charge mixture contains between about 4% and about 50% by weight of the formula IV substrate in the alkanoic acid. It is preferred to include between about 4 and about 20% by weight of alkali metal salt of acid. When acetic anhydride is used as the desiccant, it is preferably present at a ratio of about 0.05 mole to about 0.2 mole per mole of alkanoic acid.

The ratio of by-products 7,9-lactone and 11,12-lactone in the reaction mixture was used to remove trifluoroacetic acid, trifluoroacetic anhydride and leaving groups and the formation of esters (9,11-olefin) in the removal reagent. It is relatively low when a combination of potassium acetate is included as a reagent. Trifluoroacetic anhydride acts as a desiccant and is present at a rate of at least about 3% by weight, more preferably at least about 15% by weight, and most preferably about 20% by weight, based on trifluoroacetic acid and removal reagents. shall.

In addition to 7,9-lactone other impurities and by-products useful as markers on the intermediates and chromatography of the synthesis were observed at this stage of the method 1 synthesis. Novel 4,9,13-trienes of the esters of formula II (eg 7-methyl hydrogen 17-methyl-3-oxo-18-norpregna-4,9 (11), 13-tree Ene-7α, 21-dicarboxylate) was isolated by chromatography from the product solution. The amount of this compound produced appears to increase with increasing reaction time for this step of the synthesis. The compound is believed to be formed when the lactone is protonated and the resulting C17 carbonium ion facilitates the shift of the angled methyl group from the C13 position. Deprotonation of this intermediate produces 4,9,13-triene.

The novel 5-cyano of the formula II yen ester furnace - △ ^11,12 (for example 7-methyl hydrogen 17-hydroxy-3-oxo-5β- cyano -17α- frame near-11-yen -7α , 21-dicarboxylate, γ-lactone) and novel 5-cyanos (eg 7-methyl hydrogen 5-cyano-17-hydroxy-3-oxo-17α-) of the esters of formula II Pregne-11-ene-7α, 21-dicarboxylate, γ-lactone) were also isolated by chromatography from the crude product. These compounds are formed through dehydration of the remaining 5-cyano-7-acid and 5-cyano hydroxyester, respectively, and are believed to exist in the crude product solution as a result of the third step of the method 1 synthesis.

Novel C17 epimers of the esters of formula II (eg 7-methyl hydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxyl Late, γ-lactone) were also isolated by chromatography from the crude product. It is believed that the acidic conditions of the elimination reaction can lead to racemization of the C17 chiral center resulting in a 17-epimer of the ester. 17-epimer can be synthesized directly by reacting a compound of formula IV with a solution of potassium formate, formic acid and acetic anhydride and isolating the resulting 17-epimer.

Although not observed as an impurity in the crude product solution, the 11-ketone of the hydroxyester of formula V can be prepared by reacting the 11-hydroxy of the corresponding hydroxyester with a suitable oxidizing agent such as Jones reagent. Preferred 11-ketones are 7-methyl hydrogen 17-hydroxy-3,11-dioxo-17α-pregna-4-ene-7α, 21-dicarboxylate, γ-lactone.

Alternatively, the ester of formula II may be produced by removing the 11α-leaving group from the compound of formula IV and heating the solution of formula IV in an organic solvent such as DMSO, DMF or DMA.

Also according to the invention, a compound of formula IV is initially reacted with an alkenyl alkanoate, e.g. isopropenyl acetate, in the presence of an acid such as sulfonic acid toluene or an anhydrous mineral such as sulfuric acid to 3-enol ester is formed:

Alternatively, 3-enol esters can be formed by treatment of a compound of formula IV with acid anhydrides and bases such as acetic acid and sodium acetate. As another method, there is a method in which the compound of formula IV is treated with ketone in the presence of an acid to prepare a compound of formula IV (Z). The intermediate of formula IV (Z) is then reacted with alkali metal formate or acetate in the presence of formic acid or acetic acid to give the following formula IV (Y):

After Δ ^9,11 enol acetate is produced, the compound is converted into the ester of formula II by thermal decomposition of enol acetate or reaction of this compound with alkali metal alkoxide in an organic solvent, preferably an alcohol such as methanol. Can be switched. The removal reaction is highly selective for the esters of formula II, with preference for 11,12-olefins and 7,9-lactones, and this selectivity is preserved throughout the conversion to enon of enol acetate.

Preferably, the substrate of formula IV corresponds to formula IVA and the ester corresponds to formula IIA:

Formula IVA

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² , and X are as defined in formula XIIIA, and R ¹ is as defined in formula V. R ³ is preferably hydrogen.

If desired, the compound of formula II can be isolated by removing the solvent, placing the solid product in cold water, and then extracting with an organic solvent such as ethyl acetate. After appropriate washing and drying steps, the product is recovered by removing the extraction solvent. The ester is then dissolved in a solvent suitable for conversion to the product of formula I. Alternatively, the ester can be isolated by adding water to the concentrated product solution and filtering the solid product, thereby preferentially removing 7,9-lactone. The conversion of the substrate of formula II to the product of formula IA can be carried out in the manner disclosed in US Pat. No. 4,559,332, or more preferably by a novel reaction with haloacetamide as described below.

In another embodiment of the invention, the hydroxyester of formula V can be converted to the ester of formula II without isolation of the intermediate compound of formula IV. In this process, the hydroxyester is placed in an organic solvent such as methylene chloride and either an acylating agent such as methanesulfonyl chloride or a halogenating agent such as sulfyl chloride is added to this solution. The mixture is then stirred and, if halogenation is included, an HCl scavenger such as imidazole is added. This reaction is very exothermic and therefore must be carried out at a controlled rate while cooling the whole. After the base is added, the resulting mixture is warmed to a suitable temperature, such as 0 ° C. to room temperature or slightly above room temperature, and is typically reacted for 1 hour to about 4 hours. After the reaction is complete, the solvent is removed under high vacuum conditions (such as from about 24 "to about 28" Hg), preferably at about -10 ° C to about +15 ° C, more preferably at 0 ° C to about 5 ° C to concentrate the solution. And excess base is removed. The substrate is then redissolved in an organic solvent, preferably a halogenated solvent such as methylene chloride, for conversion to the esters.

The leaving group removal reagent is preferably prepared by mixing the organic acid, organic acid salt and desiccant, preferably formic acid, alkali metal formate and acetic anhydride, respectively, in a dry reactor. The addition of acetic anhydride is an exothermic reaction and as a result CO is released, the rate of addition must therefore be controlled. In order to promote the removal of water, the temperature of this reaction is preferably maintained in the range of about 60 ° C to about 90 ° C, more preferably about 65 ° C to about 75 ° C. This reagent is then added to the product solution of the compound of formula IV to effect a removal reaction. After about 4 to 8 hours the reaction mixture is preferably at a temperature of at least about 85 ° C., but at a temperature below about 95 ° C. until all volatile distillates are removed, and then for a period during which the reaction can be completed further. , Typically for about 1 to about 4 hours. After the reaction mixture is cooled and recovered by standard extraction techniques, the ester can be recovered as desired by evaporating the solvent.

In addition, the esters of formula II can be recovered from the reaction solution by an alternative process without the need for an extraction step following the removal reaction, thereby reducing costs, improving yield and / or improving productivity. In this method, the ester product is precipitated by diluting the reaction mixture with water after removing the formic acid. The product is then isolated by filtration. No extraction is necessary.

According to another method for converting the hydroxyester of formula V to the ester of formula II without isolating the compound of formula IV, after the 11α-hydroxy group of the hydroxyester of formula V is substituted with halogen, The ester of is formed in situ by dehydrohalogenation by heat. The replacement of the hydroxy group by halogen is accomplished by reaction with a sulfuryl halide, preferably sulfulyl chloride, at low temperature in the presence of a halogenated hydrogen scavenger such as imidazole. The hydroxyester is dissolved in a solvent such as tetrahydrofuran and cooled to 0 ° C. to about −70 ° C. Sulfuryl halide is added and the reaction mixture is warmed to a suitable temperature, such as room temperature, for a time sufficient to complete the removal reaction, typically for about 1 to 4 hours. The method of this embodiment combines the two steps into one, as well as halogenated reaction solutions; Acids (such as acetic acid); And the use of dry reagents (such as acetic anhydride or sodium sulfate). Moreover, the reaction does not require reflux and avoids the production of CO, a byproduct produced when acetic acid is used as a desiccant.

According to a particularly preferred embodiment of the invention, the diketone compound of formula VI can be converted to epoxymexrenone or another compound of formula I without any intermediates being isolated in purified form. According to this preferred method, the reaction solution containing hydroxyester is inhibited with a strong acid solution, cooled to ambient temperature and then extracted with a suitable extraction solvent. Advantageously, an aqueous solution of an inorganic salt, such as about 10 wt% saline solution, is added to the reaction mixture before extraction. The extract is washed and dried by azeotropic distillation to remove the remaining methanol solvent from the ketone cleavage reaction.

The resulting concentrated solution containing between about 5% and about 50% by weight of the compound of formula V is contacted with an acylating agent or alkylsulfonylating agent at low temperature to form sulfonic acid ester or dicarboxylic acid ester. After the alkylsulfonation or carboxylation reaction is complete, the reaction solution is passed through an acid exchange resin column and then over a basic exchange resin column to remove basic and acidic impurities. After each column passage, the column is washed with a suitable solvent such as methylene chloride to recover the remaining sulfonic acid or dicarboxylic acid ester from the solution. The combined eluate and wash fractions are combined and preferably reduced under vacuum to give a concentrated solution containing the sulfonic acid ester or dicarboxylic acid ester of formula IV. This concentrated solution is then contacted with a dry reagent containing an agent effective for the removal of the 11α-ester leaving group and for the separation of hydrogen to form 9,11-double bonds. Preferably, the reagent for removing the leaving group includes the formic acid / alkali metal formate / acetic anhydride dry reagent solution described above. After the reaction is complete, the reaction mixture is cooled and formic acid and / or other volatile components are removed under vacuum. The residue is cooled to ambient temperature and dried after an appropriate washing step to give a concentrated solution containing the ester of formula II. This ester can then be converted to epoxymexrenone or another compound of formula I using the methods described herein, or the methods described in US Pat. No. 4,559,332.

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 partitioned between water and a suitable organic solvent, for example ethyl acetate. The aqueous layer is then back extracted with an organic solvent and the bag extract is washed with an alkali solution, preferably an alkali metal hydroxide containing an alkali metal halide. The organic phase is preferably concentrated in vacuo to yield the ester product of formula II. The product of formula II is then placed in an organic solvent such as methylene chloride and reacted in the manner described further in US Pat. No. 4,559,332 to produce the product of formula I.

When trihaloacetonitrile is used in the epoxidation reaction, the choice of solvent is important, the solvent being found to be very preferably a halogenated solvent, and methylene chloride being particularly preferred. While solvents such as dichloroethane and chlorobenzene provide a reasonably satisfactory yield, the yield is generally better in the methylene chloride reaction medium. Solvents such as acetonitrile and ethyl acetate generally provide low yields, while reactions in solvents such as methanol or water / tetrahydrofuran can yield very little desired product.

It has also been found that according to the invention many improvements in the synthesis of epoxymexrenone can be made by using trihaloacetamide rather than trihaloacetonitrile as the peroxide activator for the epoxidation reaction. According to a particularly preferred method, epoxidation is carried out by reaction of the substrate of formula IIA with hydrogen peroxide in the presence of trichloroacetamide and a suitable buffer. Preferably, the reaction is carried out at a pH in the range of about 3 to about 7, more preferably about 5 to about 7. However, despite these considerations, successful reactions have been made outside the desired pH range.

Particularly good results are obtained with buffers containing dipotassium hydrogen phosphate and / or dipotassium hydrogen phosphate and potassium hydrogen phosphate in a ratio between about 1: 4 and about 2: 1, most preferably about Obtained as a buffer combined at a ratio of 2: 3. Borate buffers may also be used, but generally have a slower conversion rate than when using dipotassium phosphate or K ₂ HPO ₄ / KH ₂ PO ₄ mixtures. Whatever the buffer configuration, the buffer should provide a pH in the range indicated above. In addition to the overall composition of the buffer or the exact pH it can impart, it has been observed that the reaction proceeds much more effectively if at least a portion of the buffer consists of dibasic hydrogenphosphate ions. This ion can essentially act as a homogeneous catalyst in the formation of complexes comprising additives or promoters and peroxide ions, the production of which will also be essential to the overall epoxidation reaction mechanism. Therefore, the quantitative requirement for dibasic hydrophosphate (preferably derived from K ₂ HPO ₄ ) will be only a small catalytic concentration. Generally, K ₂ HPO ₄ is preferably present at least about 0.1 equivalents, such as between about 0.1 and 0.3 equivalents per substrate equivalent.

The reaction is carried out in a suitable solvent, preferably methylene chloride, or alternatively other halogenated solvents such as chlorobenzene or dichloroethane can also be used. Toluene and mixtures of toluene and acetonitrile were also found to be satisfactory. Without being bound by a particular theory, it is believed that the reaction proceeds most effectively in a two-phase system in which peroxide intermediates are formed and distributed in the low water content organic phase and then react with the substrate in the organic phase. Therefore, preferred solvents are those with low water solubility. Effective recovery from toluene is facilitated by the inclusion of other solvents such as acetonitrile.

In the conversion of the substrate of formula II to the product of formula I, toluene provides an advantage to the process because the substrate is freely soluble in toluene and the product is not. Therefore, the product precipitates during the reaction when the conversion reaches 40 to 50%, resulting in a mixture of three phases from which the product can conveniently be separated by filtration. Methanol, ethyl acetate, acetonitrile alone, THF and THF / water did not prove to be as effective as the halogenated solvent or toluene in carrying out the conversion of this step of the process.

While trichloroacetamide is a very preferred reagent, other trihaloacetamides such as trifluoroacetamide and chlorodifluoroacetamide can also be used. Trihalomethylbenzeneamide, and an arylene, alkenyl or alkynyl moiety (or withdrawn electrons of the electron withdrawing group) between the trihalomethyl group and the carbonyl group of the amide to withdraw the electrons to be transported to the amide carbonyl Other compounds with other groups may also be useful. Heptafluorobutylamide can also be used, but the result is less satisfactory. In general, the peroxide activator may correspond to the formula:

R ⁰ C (O) NH ₂

Wherein R ⁰ is at least a group having a high electron withdrawal strength (as measured by the sigma constant), such as that of a monochloromethyl group. The electron withdrawing group is preferably attached directly to the amide carbonyl for maximum effect. More specifically, the peroxide activator may correspond to the formula:

Wherein R ^P is a group which enables the transfer of the electron withdrawing group to the amide carbonyl of the electron withdrawing effect, preferably selected from arylene, alkenyl, alkynyl and-(CX ⁴ X ⁵ ) _n -moiety Become; X ¹ , X ² , X ³ , X ⁴ and X ⁵ are independently selected from halo, hydrogen, alkyl, haloalkyl and cyano and cyanoalkyl; n is 0, 1 or 2; Provided that when n is 0 at least one of X ¹ , X ² , and X ³ is halo; When R ^P is-(CX ⁴ X ⁵ ) _n -and n is 1 or 2, at least one of X ⁴ and X ⁵ is halo. If any one of X ¹ , X ² , X ³ , X ⁴ or X ⁵ is not halo, it is preferably haloalkyl, most preferably perhaloalkyl. Particularly preferred activators include those in which n is 0 and at least two of X ¹ , X ² and X ³ are halo; Or R ^P is-(CX ⁴ X ⁵ ) _n -and n is 1 or 2, at least one of X ⁴ and X ⁵ is halo, the other is halo or perhaloalkyl, X ¹ , X ² , and X ³ Are those that are halo or perhaloalkyl. Each of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is preferably Cl or F, most preferably Cl. Although mixed halides such as perhaloalkyl or perbromoalkyl and combinations thereof may also be suitable, in that case the carbon directly attached to the amide carbonyl should be substituted with at least one halo group.

Preferably, the peroxide activator is present at a ratio of at least 1 equivalent, more preferably between about 1.5 and about 2 equivalents, per equivalent of the substrate initially present. Hydrogen peroxide should be charged to the reaction in at least moderate excess, or added gradually as the epoxidation reaction proceeds. Although the reaction consumes only one to two equivalents of hydrogen peroxide per mole of substrate, it is preferred that the hydrogen peroxide is substantially overcharged relative to the substrate and activator present initially. Without limiting the invention to a particular theory, the reaction mechanism involves the formation of an activator and the addition of hydrogen peroxide anions, the formation of which is reversible to equilibrium to allow reverse reactions to occur, and thus to drive the reaction forward. It is believed that a substantial excess of hydrogen peroxide is required initially. The reaction temperature is not critical in consultation and can be effectively carried out in the range of 0 to about 100 ° C. The optimum temperature depends on the choice of solvent. In general, preferred temperatures are from about 20 ° C. to about 30 ° C., but in certain solvents such as toluene, the reaction may advantageously be performed in the range of about 60 ° C. to about 70 ° C. At about 25 ° C., the reaction typically proceeds within about 10 hours, usually within about 3 hours to about 6 hours. If desired, additional activators and hydrogen peroxide can be added at the end of the reaction cycle to achieve complete conversion of the substrate.

At the end of the reaction cycle, the aqueous phase is removed and the organic reaction solution is preferably washed to remove water soluble impurities, and then the product can be recovered by removal of the solvent. Before the solvent is removed, the reaction solution must be washed with at least a mild to moderate alkaline washing solution such as sodium carbonate. Preferably, the reaction mixture is continued with a mild reducing solution such as a weak (eg about 3% by weight) sodium sulfite solution in water to minimize product loss; Alkaline solutions, such as NaOH or KOH (preferably about 0.5 N); Acid solutions such as HCl (preferably about 1 N); And a final neutral wash containing water or saline, preferably saturated saline. Before the reaction solvent is removed, it is advantageous to add another solvent such as an organic solvent, preferably ethanol, whereby the product can be recovered by crystallization after distillation to remove more volatile reaction solvent.

The new epoxidation method utilizing trichloroacetamide or other new peroxide activators is better applied than the various methods for preparing epoxymexrenone and actually crosses olefinic double bonds in a wide range of substrates that are likely to react in the liquid phase It should be understood that it can be used to form epoxides. The reaction is particularly effective in unsaturated compounds where the olefin is tetrasubstituted and trisubstituted, ie R ^a R ^b C═CR ^c R ^{d where} R ^a to R ^d represent substituents other than hydrogen. The reaction proceeds most quickly and completely when the substrate is a cyclic compound comprising a trisubstituted double bond or a cyclic or acyclic compound containing a tetrasubstituted double bond. Exemplary such substrates for the epoxidation reaction are Δ ^9,11 -canrenone, and the following substrates:

Since the reaction proceeds more quickly and completely with triple-substituted and tetrasubstituted double bonds, such compounds (trisubstituted in compounds which may contain other double bonds when the olefinic carbon is monosubstituted or sometimes bisubstituted) It is particularly effective for selective epoxidation across double or monosubstituted double bonds.

Another non-limiting example of a general epoxidation reaction is the following epoxidation reaction:

It should also be understood that the reaction can be advantageously used in the epoxidation of monosubstituted or bisubstituted double bonds such as 11,12-olefins in various steroid substrates. However, since more highly substituted double bonds such as 9,11-olefins are preferentially epoxidized preferentially, the process of the present invention achieves high yields and high productivity in the epoxidation step of the various reaction methods described herein. It is especially effective.

The improved method is the following formula:

The following formula IB is prepared by epoxidation of:

The following formula:

By the epoxidation of the following formula IC:

It has been found that it can be applied particularly advantageously to prepare

It has been demonstrated that there are many advantages over the process of the present invention in which trichloroacetamide is used instead of trichloroacetonitrile as the oxygen transfer reagent for the epoxidation reaction. Trichloroacetamide reagent systems have low affinity for electronically deficient olefins such as α, β-unsaturated ketones. This allows for the selective epoxidation of non-bonded olefins of substrates having two types of double bonds. Incidentally, in complex substrates such as steroids, the bisubstituted olefins and the trisubstituted olefins can be distinguished by reaction. Therefore, good selectivity is observed in the epoxidation of the isomers Δ-9,11 and Δ-11,12 compounds. In this case, 9,11 epoxide is formed by minimal reaction of isomers containing Δ-11,12 double bonds. Thus, reaction yield, product profile, and final purity are substantially enhanced compared to the reaction in which trihaloacetonitrile is used. Furthermore, the substantially excess oxygen production observed by using trihaloacetonitrile has been found to be minimized using trichloroacetamide, which has been found to confer improved safety for the oxidation process. In addition, in contrast to the reaction promoted by trichloroacetonitrile, the trichloroacetamide reaction facilitates the control of the thermal profile of the reaction by minimizing the exothermic effect. The stirring effect is minimal and the reactor performance is observed to be more constant and also has the advantage over the trichloroacetonitrile method. The reaction tends to be larger than the reaction promoted with trichloroacetonitrile. Product separation and purification is simple. Bayer-villager oxidation (peroxide accelerated conversion of ketones to esters) of the carbonyl function cannot be observed as experienced when using m-chloroperoxybenzoic acid or other peracids. Reagents are cheap, readily available, and easy to manipulate.

In addition, the following compounds were observed by chromatography in the crude product from the stage of the method 1 synthesis in which the ester of formula II is converted to the compound of formula I:

(1) Novel 11α, 12α epoxides of the esters of formula II, for example 7-methyl hydrogen 11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α , 21-dicarboxylate, γ-lactone;

(2) novel 4,5: 9,11-diepoxides of the esters of formula II, such as 7-methyl hydrogen 4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo -17α-pregnan-7α, 21-dicarboxylate, γ-lactone;

(3) novel 12-ketones of the esters of formula II, for example 7-methyl hydrogen 17-hydroxy-3,12-dioxo-17α-pregna-4,9 (11) -diene-7α , 21-dicarboxylate, γ-lactone;

(4) New 9,11-dihydroxy esters of the esters of formula II, for example 7-methyl hydrogen 9α, 11β, 17-trihydroxy-3-oxo-17α-pregna-4-ene- 7α, 21-dicarboxylate, γ-lactone;

(5) Novel 12-hydroxy analogs of the esters of formula II, for example 7-methyl hydrogen 12α, 17-dihydroxy-3-oxo-17α-pregna-4,9 (11) -diene -7α, 21-dicarboxylate, γ-lactone; And

(6) novel 7-acids of compounds of formula I, for example 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone.

These compounds are utilized as markers on synthetic intermediates and / or chromatography in the preparation of compounds of formula I, in particular epoxymexrenone.

It is believed that the 11α, 12α-epoxides of the esters of formula II are formed through the impurities produced during the preceding steps where the compound of formula IV is converted to the esters of formula II. This impurity was isolated by chromatography, and is ^Δ11,12 ester. It is typically prepared using a Δ ^9,11 ester, although at a ratio of about 90:10 (Δ ^9,11 ester: Δ ^11,12 ester). During the conversion of a compound of formula II of the circle of the formula I ester △ ^11,12 yen ester is oxidized 11α, 12α - epoxide is produced.

4,5: 9,11-diepoxide of the ester of formula I is isolated by chromatography. It is believed to result from over-epoxidation of the esters. It is typically observed in crude products at levels of at least about 5% by weight or less, although the amount may vary.

The 12-ketone of the ester of formula II was isolated by chromatography. It is believed to be caused by allylic oxidation of the esters. It is typically observed at a level of about 5% by weight or less in the crude product, although the amount may vary. The level of 12-ketone detected in the crude product when trichloroacetonitrile was used as the hydrogen peroxide activator was higher than the level detected when trichloroacetamide was used as the activator.

9,11-dihydroxy of the ester of formula II was isolated by chromatography. It is typically observed at a level of about 5% by weight or less in the crude product, although the amount may vary. It is believed to result from the hydrolysis of the epoxides of formula I.

12-hydroxy of the ester of formula II was isolated by chromatography. It is typically observed in crude products at levels of 5% by weight or less, although the amount may vary. It is believed to result from hydrolysis of 11,12-epoxide, which subsequently removes 11β-hydroxy.

In addition, the compounds of formula I prepared according to the invention provide metabolites, derivatives, prodrugs and the like which have improved properties (such as improved solubility and absorption) which promote the administration and / or potency of epoxymexrenone. It can be further modified. 6-hydroxy (eg, 7-methyl hydrogen 6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregin-4-ene-7α, 21- of compounds of formula I) Dicarboxylate, γ-lactone) is a novel compound identified as a possible metabolite in rats. The 6-hydroxy metabolite is the corresponding ethyl enol ether (e.g. 7-methyl hydrogen 9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregin-4-ene-7α, 21- Dicarboxylate, γ-lactone). Ethyl enol ethers of the compounds of formula I can be prepared according to the procedures described in the literature [R.M. Weier and L.M. Hofmann, J. Med. Chem. 1977, 1304. The ethyl enol ether is then reacted with m-chloroperbenzoic acid to yield the corresponding 6-hydroxy of the compound of formula I.

It is also contemplated that monocarboxylic acid salts of epoxymexrenone, in particular potassium and sodium salts, are suitable as a surrogate for promoting the administration of the compounds of formula I to individuals instructed to administer aldosterone antagonists. Under mild basic conditions, the corresponding 17β-hydroxy-17α- (3-propionic acid) analog is obtained by selectively opening the spirolatone of the compound of formula I without hydrolyzing the C7 ester substituent. These open chain analogs are more polar than their lactone counterparts and showed shorter retention times when analyzed by reversed phase HPLC. Acidic conditions generally cause regeneration of the lactone ring.

Under more stringent conditions, spirrolactone is opened and the C7 ester is hydrolyzed to give the corresponding byproduct, 17β-hydroxy-17α- (3-propionic acid) -7-acid analog of formula I. These dicarboxylic acids show shorter retention times than monocarboxylic acids when analyzed by reversed phase HPLC. Acidic conditions (such as treatment with dilute acids such as 0.1-4 M hydrochloric acid) generally lead to regeneration of the lactone rings of the dicarboxylic acids.

The novel epoxidation process of the present invention is very useful as a critical step in the synthesis of Method 1. In a particularly preferred embodiment, the overall method of method 1 is as follows.

Is done:

Method 2

The second novel reaction method (method 2) of the present invention starts with canrenone or another substrate corresponding to formula XIII:

Formula XIII

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII above. In the first step of this process, the substrate of formula XIII is converted to the product of formula XII using the same cyanation reaction method as described above for the conversion of the substrate of formula VIII to the intermediate of formula VII:

Preferred substrates of formula XIII correspond to formula XIIIA and the enamine product corresponds to formula XIIA:

Formula XIIIA

Formula XIIA

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² , and X are as defined in formula XIIIA. R ³ is preferably hydrogen.

In the second step of method 2, the enamine of formula XII is hydrolyzed to the intermediate diketone product of formula XI using the same reaction method as described above for the conversion of the substrate of formula VIII to the intermediate of formula VII. do:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII.

Preferably, the substrate of formula XII corresponds to formula XIIA and the diketone product corresponds to formula XIA:

Formula XIIA

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² , and X are as defined in formula XIIIA. R ³ is preferably hydrogen.

Also according to reaction method 2, the diketone of formula XI is reacted with an alkali metal alkoxide to form mexrenone or another product corresponding to formula X:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII and R ¹ is as defined in formula V. The process is carried out using the reaction method substantially the same as described above for the conversion of the compound of formula VI to the compound of formula V. Preferably, the substrate of formula XI corresponds to formula XIA and the intermediate product corresponds to formula XA:

Formula XIA

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² , and X are as defined in formula XIIIA and R ¹ is as defined in formula V. R ³ is preferably hydrogen.

Mexrenone and other compounds of formula X are then 9α-hydroxylated by a novel in vivo conversion method to yield the product of formula IX:

Formula IX

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII and R ¹ is as defined in formula V. Among the organisms that can be used in this hydroxylation step are the following: Nocardia conicruria ATCC 31548, N. aurentia ATCC 12674, Corynespora cassicola ( Corynespora cassiicola) ATCC 16718, Streptomyces dihydro Scorpion kusu (Streptomyces hydroscopicus) ATCC 27438, Mortierella Isabel Lina (Mortierella isabellina) ATCC 42613, view Calabria bassi Ana (Beauvria bassiana) ATCC 7519, Penny silryum greener furo genum (Penicillum purpurogenum ATCC 46581, Hypomyces chrysospermus IMI 109891, Thamnostylum piriforme ATCC 8992, Cunninghamella blakesleeana ATCC 8688a, Equine Gammella C. echinulata ATCC 3655, C. elegans ATCC 9245, Trichotesium Rose thecium roseum ATCC 12543, Epicoccum humicola ATCC 12722, Saccharopolyspora eythrae ATCC 11635, Bubria basiaa ATCC 13144, Arthrobacter simplex , Bacterium cyclo Bacterium cyclooxydans ATCC 12673, Cylindrocarpon radicicola ATCC 11011, N. aurentia ATCC 12674, N. restrictus ATCC 14887 Pseudomonas testosteroni ATCC 11996, Rhodococcus equi ATCC 21329, Mycobacterium fortuitum NRRL B8119, and Rhodococcus rhodochrous reactions were substantially ATCC 19150. 1 and 2 in the manner described above. The method of FIG. 1 is particularly preferred.

Growth media useful for in vivo conversion preferably include between about 0.05% and about 5% available nitrogen; Between about 0.5 weight% and about 5 weight% glucose; Yeast derivatives between about 0.25 weight% and about 2.5 weight%; And between about 0.05 weight% and about 0.5 weight% usable phosphorus. Particularly preferred growth media include the following:

Soybean meal: between about 0.5% by weight and about 3% by weight glucose; Between about 0.1 wt% and about 1 wt% soybean mill; Between about 0.05 weight% and about 0.5 weight% alkali metal halide; Between about 0.05% and about 0.5% by weight yeast derivatives, such as self-dissolved yeast or yeast extract; Between about 0.05 wt% and about 0.5 wt% phosphate, such as K ₂ HPO ₄ ; pH = 7;

Peptone-yeast extract-glucose: between about 0.2 weight% and about 2 weight% peptone; Yeast extract between about 0.05% and about 0.5% by weight; And between about 2 wt% and about 5 wt% glucose;

Muller-Hinton: Beef infusion between about 10% and about 40% by weight; Between about 0.35 wt% and about 8.75 wt% casamino acid; Starch between about 0.15% and about 0.7% by weight.

Fungi can grow in soybean wheat or peptone nutrients, while actinomycetes and eubacteria are added to soybean wheat (0.5% to 1% by weight of carboxylic acid salts, such as sodium formate, for in vivo conversion). ) Or Müller-Hinton gravy.

The preparation of ll [beta] -hydroxymexrenone from mexrenone by fermentation is discussed in Example 19B below. Similar bioconversion methods can be used to prepare other starting materials and intermediates. Example 19A describes the bioconversion of androstenedione to 11β-hydroxyandrostenedione. Example 19C describes bioconversion of mexrenone to 11α-hydroxymexrenone, Δ ^1,2 -mexrenone, 6β-hydroxymexrenone, 12β-hydroxymexrenone, and 9α-hydroxymexrenone do. Example 19D describes the bioconversion of ^canrenone to Δ ^9,11 -mexrenone.

The product of formula IX is a novel compound, which can be separated by filtration, washed with a suitable organic solvent such as ethyl acetate, and recrystallized from the same or similar solvent. They have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Preferably, the compound of formula IX is -AA- and -BB- is -CH ₂ -CH _2- , R ³ is hydrogen, lower alkyl or lower alkoxy, and R ⁸ and R ⁹ together are 20-spir Corresponds to the formula IXA constituting the roxane ring:

(Formula XXXIII)

In the next step of the method 2 synthesis, the product of formula IX is reacted with a dehydration reagent (suitable dehydration reagents such as PhSOCl or ClSO ₃ H are known to those skilled in the art) to yield a compound of formula II:

Formula II

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII and R ¹ is as defined in formula V.

Preferably, the substrate of formula IX corresponds to formula IXA and the intermediate product corresponds to formula IIA:

Formula IIA

In the above formulas, -AA-, -BB-, R ³ , Y ¹ , Y ² , and X are as defined in formula XIIIA and R ¹ is as defined in formula V. R ³ is preferably hydrogen.

In the final step of this synthesis process, the product of formula II is prepared by epoxidation according to the method disclosed in US Pat. No. 4,559,332; Or is preferably converted to the product of formula I by the novel epoxidation process of the invention as described herein.

In a particularly preferred embodiment, the overall method of method 2 proceeds as follows:

Method 3

In this case, the synthesis begins with a substrate corresponding to formula XX:

Wherein -AA- and R ³ are as defined in formula XIII and -BB- are as defined in formula XIII, provided that neither R ⁶ nor R ⁷ is fused to the D ring at position 16,17 Not part of the ring, R ²⁶ is lower alkyl, preferably methyl. R ³ is preferably hydrogen.

Reaction of the substrate of formula XX with the sulfonium illide yields an epoxide intermediate corresponding to formula XIX:

Wherein -AA-, -BB-, R ³ and R ²⁶ are as defined in formula XX. R ³ is preferably hydrogen.

In the next step of synthesis method 3, the intermediate of formula XIX is further converted to an intermediate of formula XVIII:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. R ³ is preferably hydrogen. In this step, the substrate of formula XIX is converted to the intermediate of formula XVIII by reaction with NaCH (COOEt) _{2 in} a solvent in the presence of a base.

The compound of formula XVIII is exposed to heat, water and an alkali metal halide to yield a decarboxylated intermediate compound corresponding to formula XVII:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. R ³ is preferably hydrogen. The conversion of the compound of formula XX to the compound of formula XVII essentially corresponds to the method described in US Pat. Nos. 3,897,417, 3,413,288 and 3,300,489. Although the substrates are different, the reagents, mechanisms and conditions for the introduction of the 17-spirolactone moiety are essentially identical.

The reaction of the intermediate of formula XVII with the dehydration reagent yields the intermediate of formula XVI:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. R ³ is preferably hydrogen.

Typically useful dehydration reagents are dichlorodicyanobenzoquinone (DDQ) and chloranyl (2,3,5,6-tetrachloro-p-benzoquinone). Alternatively, dehydration may be by sequential halogenation at the 6-position carbon followed by dehydrohalogenation reaction.

The intermediate of formula XVI is then converted to the enamine of formula XVB:

Formula XVB

Wherein -AA-, -BB- and R ³ are as defined in formula XX. R ³ is preferably hydrogen. The conversion is essentially by cyanation in the manner described above for the conversion of the 11α-hydroxy compound of formula VIII to the enamine of formula VII. Typically, the cyanide ion source may be an alkali metal cyanide. The base is preferably pyrrolidone and / or tetramethylguanidine. Methanol solvents may also be used.

The product of formula XVB is a novel compound, which can be isolated by chromatography. These and other novel compounds of formula XV have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Compounds of formula XV correspond to the following structural formula:

Formula XV

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII. In the most preferred compounds of formulas XV and XVB, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

According to the hydrolysis described above to prepare the diketone compound of formula VI, the enamine of formula XVB can be converted to the diketone of formula XIVB:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. R ³ is preferably hydrogen. Particularly preferred for the synthesis of epoxymexrenone are the compounds of formula XIV which fall within the scope of formula XIVB as described below.

The product of formula XIVB is a novel compound, which can be isolated by precipitation. These and other novel compounds of formula XIV have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Compounds of formula XIV correspond to formula XIV:

Formula XIV

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII. In the most preferred compounds of formulas XIV and XIVB, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

The compounds of formula XIVB are essentially converted to the compounds of formula XXXI using the method described above to convert the diketone of formula VI to the hydroxyester of formula V. In this case, it is necessary for the intermediate of formula XXXI to be isolated before continuing conversion to the product of formula XXXII:

In the above formulas, -AA-, -BB- and R ³ are as defined in formula XX and R ¹ is as defined in formula V. R ³ is preferably hydrogen. Preferred compounds of formula XXXI are those belonging to formula IIA. Compounds of formula XXXI are converted to compounds of formula XXXII using the method described herein or the method of US Pat. No. 4,559,332.

Preferred compounds of formula XIV are 4'S (4'α), 7'α-1 ', 2', 3 ', 4,4', 5,5 ', 6', 7 ', 8',

10 ', 12', 13 ', 14', 15 ', 16'-hexadecahydro-10β-, 13'β-dimethyl-3', 5,20'-trioxospyro [furan-2 (3H), 17'β- [4,7] methano [17H] -cyclopenta [a] phenanthrene] 5'-carbonitrile; Preferred compounds of the formula XV are 5'R (5'α), 7'β-20'-amino-1 ', 2', 3 ', 4,5,6', 7 ', 8', 10 ', 12 ', 13', 14 ', 15', 16'-tetradecahydro-10'α, 13'α-dimethyl-3 ', 5-dioxospyro [furan-2 (3H), 17'α (5' H)-[7,4] metheno [4H] -cyclopenta [a] phenanthrene] -5'-carbonitrile. In a particularly preferred embodiment, the overall method of method 3 proceeds as follows:

Method 4

The first three steps of Method 4 are the same as the first three steps of Method 3. In other words, the preparation of the intermediate of formula XVII starts with the compound corresponding to formula XX.

The intermediate of formula XVII is then epoxidized using, for example, the method of US Pat. No. 4,559,332 to give a compound of formula XXIV:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. However, in a particularly preferred embodiment of the invention, the substrate of formula XVII is a peroxide activator of the amide type, most preferably according to the method described above in method 1 for converting the ester of formula II to the product of formula I Preferably, it is epoxidized across 9,11-double bonds using an oxidation reagent containing trichloroacetamide. The conditions for this reaction and the ratio of reagents are substantially as described for the conversion of the esters of formula II to epoxymexrenone. Particularly preferred compounds of formula XXIV are those in which -AA- and -BB- are as defined in formula XIII and R ³ is hydrogen.

It has been found that epoxidation of the substrate of formula XVII can also be achieved in very good yields using peracids such as m-chloroperoxybenzoic acid. However, trichloroacetamide reagents provide superior results in minimizing the formation of Bayer-Villager oxidation by-products. The latter byproduct can be removed, but it needs to be titrated from a solvent such as ethyl acetate and then crystallized from another solvent such as methylene chloride. The epoxy compound of formula XXIV is dehydrogenated by reaction with a dehydrogenating (oxidizing) agent such as DDQ or chloranyl to produce a double bond between 6-carbon and 7-carbon, or brominated / dehydrobrominated (or other halogenated) / Dehydrohalogenation) sequence to produce another novel intermediate of formula XXIII:

Formula XXIII

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XX.

Particularly preferred compounds of formula XXIII are those wherein -AA- and -BB- are as defined in formula XIII and R ³ is hydrogen.

While direct oxidation is effective for the formation of the product of formula XXIII, the yield is generally low. Therefore, the oxidation is preferably carried out in two stages, in which the substrate of formula XXIV is halogenated at the C-6 position and then dehydrohalogenated to 6,7-olefin. Halogenation preferably takes place using N-halo organic reagents such as, for example, N-bromosuccinamide. Bromination is carried out in a suitable solvent such as acetonitrile in the presence of a halogenation promoter such as benzoyl peroxide. The reaction proceeds effectively in a solvent such as carbon tetrachloride, acetonitrile or mixtures thereof at a temperature in the range from about 50 ° C. to about 100 ° C., conveniently at ambient reflux. However, typically a reaction of 4 to 10 hours is required for the completion of the reaction. The reaction solvent is removed and the residue is placed in a solvent which does not mix with water, such as ethyl acetate. The resulting solution is washed sequentially with mild alkaline solution (such as alkali metal bicarbonate) and water, or preferably with saturated brine, to minimize product loss, after which the solvent is removed and the residue is suitable for the dehydrohalogenation reaction. In another solvent (such as dimethylformamide).

Suitable dehydrohalogenation reagents, such as 1,4-diazabicyclo [2,2,2] octane (DABCO), are added to the solution together with alkali metal halides such as LiBr, and the solution is brought to a suitable reaction temperature, such as from 60 ° C. to 80 ° C. The reaction is heated to < RTI ID = 0.0 > C, < / RTI > for several hours, typically 4 to 15 hours, until the dehydrohalogenation reaction is complete. Additional dehydrohalogenation reagents may be added during the reaction cycle to induce completion of the reaction, if desired. The product of formula XXIII can then be recovered, for example, by adding water to the precipitate, separating the product by filtration and preferably washing with an additional amount of water. The product is preferably recrystallized from, for example, dimethylformamide.

The product of formula XXIII, such as 9,11-epoxycanrenone, is a novel compound and can be isolated by extraction / crystallization. This product has substantial value as an intermediate for preparing compounds of formula I, in particular formula IA. They can be used, for example, as substrates for the preparation of compounds of formula XXII.

Subsequently, using the method described above for the preparation of the compound of formula VII, the compound of formula XXIII is reacted with cyanide ions to produce a novel epoxyenamine compound corresponding to formula XXII:

Formula XXII

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XX. Particularly preferred compounds of formula XXII are those in which -AA- and -BB- are as defined in formula XIII and R ³ is hydrogen.

The product of formula XXII is a novel compound, which can be isolated by precipitation and filtration. They have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. In the most preferred compounds of formula XXII, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

Subsequently, using the method described above to prepare compounds of Formula VI, the epoxyenamine compounds of Formula XXII are converted to novel epoxydiketone compounds of Formula XXI:

Formula XXI

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula XIII. In the most preferred compounds of formula XXI, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

The product of formula XXI is a novel compound, which can be isolated by precipitation and filtration. They have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Particularly preferred compounds of formula XXI are those in which -AA- and -BB- are as defined in formula XIII. In the most preferred compounds of formula XXI, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

Using the process described above to prepare substantially the hydroxyester compounds of formula V from the diketone compounds of formula VI, the epoxydiketone compounds of formula XXI are converted into compounds of formula XXXII:

(Formula XXXII)

Wherein -AA-, -BB- and R ³ are as defined in formula XX and R ¹ is as defined in formula V.

As in the conversion of the diketone of formula V to the hydroxyester of formula VI, the 5-β-cyano-7-ester intermediate is also formed in the conversion of the epoxydiketone of formula XXI to the compound of formula XXXII. In both series the 5-β-cyano-7-ester intermediate can be isolated by treating the corresponding diketone with an alcohol such as methanol in the presence of a base such as triethylamine. Preferably, the intermediate is prepared by refluxing a mixture of diketones for about 4 to about 16 hours in alcohol, such as methanol, containing about 0.1 to 2 equivalents of triethylamine per mole of diketone. The product is isolated in pure form by cooling the mixture to about 25 ° C. and then filtering. The isolated intermediate can be converted into compounds of formula XXXII when treated with a base such as an alkali metal alkoxide in a solvent, preferably an alcohol such as methanol. The use of alkoxides in alcohols results in an equilibrium mixture similar to that formed when the corresponding diketone of formula XXI is treated under the same conditions.

In addition, 7β-esters of the compound of formula XXXII (eg 7-methyl hydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7β, 21-dicarboxyl Rate, γ-lactone) was observed by chromatography of the crude product of the last step of method 4. The alkoxide and / or cyanide in the solution react to convert the 7α-ester into an epimer mixture of 7α-ester and 7β-ester epimer. Pure 7β-esters can be isolated from epimer mixtures by selective crystallization.

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

In a particularly preferred embodiment, the overall method of method 4 proceeds as follows:

Method 5

The method of method 5 starts with a substrate corresponding to formula XXIX:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. Under conditions similar to those described in Example 19B, the following microorganisms can be subjected to 9α-hydroxylation of a compound of formula XXXV (such as androstenedione) to a compound of formula XXIX:

(Wherein -AA-, -BB- and R ³ are as defined in formula XIII)

Asperigilus niger ATCC 16888 and 26693, Corynespora cassiicola ATCC 16718, Curvularia clavata ATCC 22921, Mycobacterium fortuitum , NRRL B 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 formula XXIX is reacted with trimethylorthoformate to be converted to the product of formula XXVIII:

Wherein -AA-, -BB- and R ³ are as defined in formula XX. Following formation of the compound of formula XXVIII, this compound is converted to the compound of formula XXVII using the method described above for the conversion of the substrate of formula XX to formula XVII:

Wherein -AA-, -BB- and R ³ are as defined in formula XX and R ^x is one of the common hydroxy protecting groups. Alternatively, the C9 α-hydroxy group can be protected in the preceding step of this synthesis method if protection at that step is required, ie C9 hydroxy of the compound of formula XXVIII or C9 hydroxy of the compound of formula XXIX It may be protected by any one of the phosphorus hydroxy protecting groups.

Using the method described above for the preparation of compounds of formula XVI, the compounds of formula XXVII are oxidized to yield novel compounds corresponding to formula XXVI:

Formula XXVI

Wherein -AA-, -BB- and R ³ are as defined in formula XX. Particularly preferred compounds of formulas XXIX, XXVIII, XXVII and XXVI are those wherein -AA- and -BB- are as defined in formula XIII and R ³ is hydrogen.

The products of formula XXVI are novel compounds, which can be isolated by precipitation / filtration. They have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Particularly preferred compounds of formula XXVI are those wherein -AA- and -BB- are as defined in formula XIII and R ³ is hydrogen. In the most preferred compounds of formula XXVI, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

Using the method defined above for cyanation of the compounds of formula VIII, the novel intermediate of formula XXVI is converted to the novel 9-hydroxyenamine intermediate of formula XXV:

Formula XXV

Wherein -AA-, -BB- and R ³ are as defined in formula XX.

The products of formula XXV are novel compounds, which can be isolated by precipitation / filtration. They have substantial value as intermediates for the preparation of compounds of formula I, in particular formula IA. Particularly preferred compounds of formula XXV are those wherein -AA- and -BB- are as defined in formula XIII and R ³ is hydrogen. In the most preferred compounds of formula XXV, -AA- and -BB- are -CH ₂ -CH ₂ -and R ³ is hydrogen.

Essentially using the conditions described above for the preparation of the diketone compounds of formula VI, the 9-hydroxyenamine intermediate of formula XXV is converted to the diketone compound of formula XIVB. In this case, it should be noted that the reaction is effective against simultaneous hydrolysis of the enamine structure and dehydration at the 9,11 position to introduce the 9,11 double bond. The compound of formula XIV is then converted to the compound of formula XXXI and again to the compound of formula XIII using the same steps as detailed above in method 3.

Preferred compounds of formula XIV are 4'S (4'α), 7'α-1 ', 2', 3 ', 4,4', 5,5 ', 6', 7 ', 8',

10 ', 12', 13 ', 14', 15 ', 16'-hexadecahydro-10β-, 13'β-dimethyl-3', 5,20'-trioxospyro [furan-2 (3H), 17'β- [4,7] methano [17H] -cyclopenta [a] phenanthrene] 5'-carbonitrile; Preferred compounds of the formula XXV are 5'R (5'α), 7'β-20'-aminohexadecahydro-9'β-hydroxy-10'a, 13'α-dimethyl-3 ', 5-di Oxospyro [furan-2 (3H), 17'α (5'H)-[7,4] metheno [4H] cyclopenta [a] phenanthrene] -5'-carbonitrile; Preferred compounds of formula XXVI are 9α, 17α-dihydroxy-3-oxopregna-4,6-diene-21-carboxylic acid, γ-lactone; Preferred compounds of the formula XXVII are 9α, 17α-dihydroxy-3-oxopregan-4-ene-21-carboxylic acid, γ-lactone.

In a particularly preferred embodiment, the overall method of method 5 proceeds as follows:

Method 6

Method 6 comprises an epoxymexrenone, wherein an intermediate corresponding to formula XXXVI or an equivalent 11β-hydroxy isomer thereof is produced starting from 11α or 11β-hydroxylation of androstenedione or another compound of formula XXXV Provides an advantageous method for preparing other compounds corresponding to I:

(Formula XXXV)

Wherein -AA-, -BB- and R ³ are as defined in formula XIII.

Except for the selectivity to the substrate, the method of performing 11α-hydroxylation is essentially the same as described above for Method 1. The following microorganisms can perform 11α-hydroxylation of androstenedione or other compounds of formula XXXV:

Absidia glauca ATCC 22752, Aspergillus flavipes ATCC 1030, A. foetidus ATCC 10254, A. fumigatus ) ATCC 26934, A. ochraceus NRRL 405 (ATCC 18500), A. niger ATCC 11394, A. nidulans ATCC 11267, Beauveria bassiana ATCC 7159, Fusarium oxysporum ATCC 7601, F. oxysporum cepae ATCC 11171 , F. lini ATCC IFO 7156 , jibe Pasteurella puji Corey (Gibberella fujikori) ATCC 14842, hypo Mrs. Cri source FER mousse (Hypomyces chyrsospermus) IMI 109891, Mycobacterium poreutuyi Tomb (Mycobacterium fortuitum) NRRL B8119, Penny silryum wave turum (Penicillum patulum) ATCC 24550, peak North Pori Umm species (Pycnosporium spp.) ATCC 12231, Rizzo crispus Ari juice (Rhizopus arrhizus) ATCC 11145, Saka in Indianapolis Fora Erie Tribe ah (Saccharopolyspora erythraea) ATCC 11635, tamno still room flutes Fort Tome (Thamnostylum pirifprme) ATCC 8992, Rizzo R. oryzae ATCC 11145, R. stolonifer ATCC 6227b, and Tricothecium roseum ATCC 12519 and ATCC 8685 .

Microorganisms capable of carrying out 11β-hydroxylation of androstendeon or other compounds of formula XXXV are as follows:

Aspergillus fumigatus (A. fumigatus) ATCC 26934, Aspergillus nigeo (A. niger) ATCC 16888 and ATCC 26693, the epi-party kokum Duck (Epicoccum oryzae) ATCC 7156, Mercure Patria ing Luna Other (Curvularia lunata ) ATCC 12017, Cunninghamella blakesleeana ATCC 8688a, and Pithomyces atro-olivaceous IFO 6651 .

11α-hydroxyandrost-4-ene-3,17-dione, or another compound of formula XXXVI, is then reacted with an etherification reagent such as trialkyl orthoformate in the presence of an acid catalyst 101) is converted to 11α-hydroxy-3,4-enol ether:

Formula 101

Wherein -AA-, -BB- and R ³ are as defined in formula XIII and R ¹¹ is methyl or other lower alkyl (C ₁ to C ₄ ). To carry out this conversion reaction, the 11α-hydroxy substrate is mixed with an acid such as benzene sulfonic acid hydrate or toluene sulfonic acid hydrate and acidified by dissolution in a lower alcohol solvent, preferably ethanol. Trialkyl orthoformate, preferably triethyl orthoformate, is introduced gradually over 5 to 40 minutes while maintaining the mixture at low temperature, preferably between 0 ° C and about 15 ° C. The mixture is then warmed up and the reaction is carried out between 20 ° C and about 60 ° C. Preferably the reaction is carried out at 30 ° C. to 50 ° C. for 1 to 3 hours and then heated to reflux for an additional period, typically 2 to 6 hours to complete the reaction. The reaction mixture is preferably cooled to 0 to 15 ° C., more preferably to about 5 ° C. and the solvent is removed under vacuum.

Using the same reaction method as described in Method 3 for the conversion of the compound of formula XX to the compound of formula XVII, the 17-spirolactone portion of formula XXXIII is introduced into the compound of formula 101. For example, the substrate of Formula 101 may be reacted with sulfonium illide in a suitable solvent such as DMSO in the presence of a base such as an alkali metal hydroxide to produce an intermediate corresponding to Formula 102:

Formula 102

Wherein -AA-, R ³ , R ¹¹ and -BB- are as defined in equation 101. The intermediate of formula 102 is reacted with malonic acid diester in the presence of an alkali metal alkoxide in the following step to form a 5-membered spirolactone ring and yield the intermediate of formula 103:

(Formula 103)

Wherein -AA-, R ³ , R ¹¹ and -BB- are as defined in equation 102 and R ¹² is C ₁ to C ₄ alkyl, preferably ethyl. Finally, the compound of formula 103 in a suitable solvent such as dimethylformamide is heated in the presence of an alkali metal halide, and the alkoxycarbonyl moiety is separated to yield an intermediate of formula 104:

Formula 104

Wherein -AA-, R ³ , R ¹¹ and -BB- are as defined in equation 102.

The 3,4-enol ether compound of formula 104 is then converted into a compound of formula XXIII, ie a compound of formula VIII wherein R ⁸ and R ⁹ together form a portion of formula XXXIII. This oxidation step is carried out in essentially the same manner as the oxidation step for the conversion of the compound of formula XXIV to the intermediate of formula XXIII in the synthesis of Method 4. Direct oxidation can be accomplished using reagents such as, for example, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or tetrachlorobenzoquinone (chloranyl), or preferably both The oxidation of the step is first brominated with an N-halo bromination agent such as, for example, N-bromosuccinamide (NBS) or 1,3-dibromo-5,5-dimethyl hydantoin (DBDMH), followed by LiBr By dehydrobromination with a base, for example DABCO, in the presence and heating. If NBS is used for bromination, an acid must also be used to convert 3-enol ethers to enons. DBDMH, which is more ionic than free radical bromination reagents, is effective on its own in converting bromination and enol ethers to enons.

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

Each of the intermediates of formulas 101, 102, 103 and 104 are novel compounds that have substantial value as intermediates to the epoxymexrenones of formulas IA and I or other compounds. In each of the compounds of the formulas 101, 102, 103 and 104, -AA- and -BB- are preferably -CH ₂ -CH ₂ -and R ³ is hydrogen, lower alkyl or lower alkoxy. R ³ is preferably hydrogen. Most preferred compound of formula 101 is 3-ethoxy-11α-hydroxyandrost-3,5-diene-17-one; Preferred compound of formula 102 is 3-ethoxyspiro [androst-3,5-diene-17β, 2'-oxirane] -11α-ol; Preferred compounds of formula 103 are ethyl hydrogen 3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21,21-dicarboxylate, gamma-lactone; Preferred compounds of formula 104 are 3-ethoxy-11α-17α-dihydroxypregna-3,5-diene-21-carboxylic acid, gamma-lactone.

In a particularly preferred embodiment, the overall method of method 6 proceeds as follows:

It is believed that the epoxymexrenone and other compounds corresponding to formula I can likewise be prepared from 11β-hydroxylated 11β-hydroxyandrostenedione or other compounds of formula XXXV. In other words, the epoxymexrenone and other compounds corresponding to formula I may be prepared using an α-hydroxylated substrate or a corresponding β-hydroxylated substrate of formula XXXV following the general method described in Method 6. Can be.

Method 7

Method 7 provides a process for the synthesis of epoxymexrenone and other compounds of formula I using a starting substrate comprising β-sitosterol, cholesterol, stigmasterol or other compounds of formula XXXVII:

Wherein -AA-, R ³ , and -BB- are as defined in formula XIII; DD is -CH ₂ -CH ₂ -or -CH = CH-; Each of R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ is independently selected from hydrogen or C ₁ to C ₄ alkyl. R ³ is preferably hydrogen.

In the first step of the synthesis, 11α-hydroxyandrostendione or other compound of formula XXXVI is prepared by bioconversion of a compound of formula XXXVII. The bioconversion method is carried out substantially according to the method described above for 11α-hydroxylation of canrenone (or other substrate of formula XIII).

In the synthesis of llα-hydroxyandrostenedione, 4-androsten-3,17-dione is initially prepared by bioconversion of the compound of formula XXXVII. This initial bioconversion can be performed in the manner described in US Pat. No. 3,759,791. 4-Androsten-3,17-dione is then converted to 11α-hydroxyandrostenedione substantially following the method described above for 11α-hydroxylation of canrenone (or other substrate of formula XIII).

The remainder of the synthesis of Method 7 is identical to Method 6. In a particularly preferred embodiment the overall method of method 7 proceeds as follows:

Epoxymexrenone and other compounds of formula I are methods where the product of bioconversion of β-cytosterol or other compounds of formula XXXVIII is 11β-hydroxyandrostendione or other compounds of formula XXXV having β-hydroxylation It is believed that it can be prepared according to the general method described in 7. In other words, the epoxymexrenone and other compounds corresponding to Formula I are those wherein the bioconversion of β-sitosterol or other compounds of Formula XXXVIII results in an α-hydroxylated substrate of Formula XXXV or a corresponding β-hydroxylated substrate. It can be prepared according to the general method described in Method 7 when inducing the production of either.

Method 8

A fairly complex problem in the synthesis of epoxymexrenone and related compounds is the need to stereoselectively introduce α-alkoxycarbonyl substituents at 7-carbon without causing unwanted modifications to other sites of the steroid structure. According to the present invention, an effective synthetic route for introducing 7a-alkoxycarbonyl substituents has been found to include the following steps: (i) initial cyanation at 7-carbon of the steroid, (ii) of 7-cyano steroid Formation of a mixture of 7α-carboxylic acids and 7β-carboxylic acids due to hydrolysis, (iii) formation of 5,7-lactone steroids from 7α-carboxylic acid steroids, and (iv) 7β from 5,7-lactone steroids Separation of carboxylic acids. Base-mediated open reaction of 5,7-lactone steroids with alkylating agents yields the desired 7α-alkoxycarbonyl steroids.

Thus, the method of method 8 generally comprises reacting a 3-keto-4,5-dihydro-5,7-lactone steroid substrate with an alkylating agent in the presence of a base, with 3-keto-7α-alkoxycarbonyl substitution It relates to a method for producing Δ ^4,5 -steroid. The lactone substrate is substituted with keto at 3-carbon and further includes the following moieties:

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

According to the present invention, an advantageous method for the preparation of the 4,5-dihydro-5,7-lactone steroid compounds described above has also been found. In this method, the 3-keto - △ ^4,5 -7α--cyano substituted steroid substrate is converted to the 7-carboxylic acid, the acid is continuously reacted with the tree in an acidified lower alcohol solvent, alkyl ortho formates 5 , 7-lactone is produced. Reaction with ortho formic acid esters also converts the 3-keto group to 3-acyclic or cyclic ketal 5,7-lactone (under which lactones are initially formed). Preferably 3-ketal 5,7-lactone is 3-dialkyl ketal 5,7-lactone. More preferred is that the alkyl portion of the alcohol solvent is identical to the alkyl portion of the orthoformate alkoxy group (most preferably all methyl): since the alkoxy portion of the ketal can be derived from orthoformate or alcohol; Mixed ketals are undesirable; This is because 3-dimethoxy is preferred. When the ketal is ethylene ketal, the alkyl portion of the alcohol solvent does not necessarily have to be the same as the alkyl portion of the orthoformate alkoxy group. 3-Ketal-5,7-lactone is readily hydrolyzed to 3-keto-5,7-lactone, a crystalline compound that can be easily purified. Since only 7α-carboxylic acid can advance the lactonation reaction, complete stereospecificity is achieved. 7β-acid can then be removed from the reaction mixture in its salt form, for example by treating the 7β-acid with a mild base such as sodium bicarbonate.

7-cyano substrates for the formation of 5,7-lactone can be prepared in a known manner. For example, an unsubstituted substrate at 7-carbon may be reacted in a slightly excess, preferably weakly acidic solution containing about 1.05 to about 1.25 equivalents of cyanide ions per equivalent of substrate and a water / DMSO solvent mixture. Can be. Preferably, the reaction mixture contains about 1 equivalent of acetic acid per equivalent of carboxylic acid, such as a substrate. Both 7α- and 7β-CN isomers are formed, mainly 7α-isomers. The 7α-cyano steroid can be recovered in a conventional manner. Other methods known in the art are also useful for this auxiliary preparation.

Generally according to method 8, 5,7-lactone is substituted with a keto, R ⁸ or R ⁹ at the 17-position (R ⁸ and R ⁹ are as described above) and an aliphatic, olefin, epoxide or hydroxy substitution Can be formed from 7-carboxy intermediates having the form in C-9 and C-11 (the compound itself is prepared by hydrolyzing the 7-cyano intermediate), ie:

Wherein -AA-, -BB- and R ³ are as defined above, R ⁸⁰ and R ⁹⁰ are the same as R ⁸ and R ⁹ , or R ⁸⁰ and R ⁹⁰ together form a keto group, R ¹⁸ is as described below for Method 9, and -EE- is selected from the following groups:

Formula XLIII

Formula XLIV

Formula XLV

(Formula XLVI)

Formula XLVII

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

In each of the formulas XL, XLI, XLII and XLVIII, R ⁸⁰ and R ⁹⁰ together preferably constitute a keto or the following formula XXXIV, most preferably R ⁸⁰ and R ⁹⁰ together constitute a formula XXXIII:

(Formula XXXIV)

Wherein Y ¹ , Y ² , X and C (17) are as defined above)

(Formula XXXIII)

R ³ is preferably hydrogen, R ¹ is preferably methoxycarbonyl, and -AA- and -BB- are each preferably -CH ₂ -CH _2- . The reaction can also be carried out using a protected 3-keto group by converting the 3-keto group to ether or keral and maintaining it in the form of either ether or ketal throughout the reaction. Another method of method 8 involves using various intermediates that fall within the scope of formulas XLI and XLII as mentioned above.

In method 8 the reagent for forming 5,7-lactone from 3-keto-Δ ^4,5-7 -carboxylic acid is trialkyl orthoformate, which is the 3- of 11α-hydroxyandrostenedione in method 6 It should be noted that this is the same reagent used for the conversion to enol ether-3,5-diene-11α-hydroxy intermediate 101, and the route of reaction of Method 8 is believed to depend on the substituents on C-7. Reaction with orthoformate in the presence of H ⁺ results in the formation of intermediate carbonium ions having a carboxyl at C-7 and an equilibrium positive charge between C-3 and C-5. When the protons are lost, the compound of formula 101 is produced with C-3 carbonium ions, while lactone is produced with C-5 carbonium ions. As hydrogen in C-7, 3,5-diene-3-alkoxy (enol ether) is preferred because of double bond inclusions. Using the 7α-CO ₂ substituent in C-7, C-5 carbonium ions are captured by carboxy and 5,7-lactone is formed. At this point the 3-keto group is preferably converted to ketal, whereby the reaction is complete.

Embodiments of the preferred method 8 are described in methods 9 and 10.

Method 9

Method 9 starts with the same substrate as in method 4, ie the compound of formula XX. This substrate is first oxidized to the compound of formula B:

Wherein -AA-, R ³ and -BB- are as defined in formula XIII.

The oxidation reaction is carried out according to any of the reaction methods described above for the conversion of the compound of formula XXIV to the intermediate of formula XXIII in the synthesis of method 4. Using the method of Method 8, the compound of Formula B is converted to the 7-cyano intermediate of Formula C:

Wherein -AA-, R ³ and -BB- are as defined in formula XIII.

Next, the compound of formula C is converted to 5,7-lactone of formula D using the trialkyl orthoformate reagent used previously in method 6:

Wherein -AA-, R ³ and -BB- are as defined in formula XIII and R ¹⁷ is C ₁ -C ₄ alkyl. The 5,7-lactone of formula D is unreacted 7- by reacting with the bicarbonate to remove the acid, thereby establishing the desired C-7 stereochemistry and preventing epimerization in subsequent reactions carried out under basic conditions. It is easily separated from β-COOH. As described in Method 8, esterification of lactones following reaction with alkyl halides results in the ester intermediates of formula II.

Continuing the synthesis of method 9, the compound of formula D is converted to the compound of formula II. Using a 3-keto group protected by conversion to ketal, the 20-spiroxane ring of formula XXXIII is optionally introduced at the 17-position following the reaction method described for methods 3 and 6, thereby producing a compound of formula E do:

Since 3-ketone is protected, optimal hydrolysis conditions can be selected to attack 17-ketone regardless of the formation of by-products through reaction at the 3-position. The 3-ketal compound of formula E is represented by the following formula F:

 After hydrolysis to the 3-keto group structure of, the latter intermediate is reacted with alkyl iodide in the presence of a base, in accordance with the conditions of Method 8 to give an ester intermediate of formula II. Finally, the latter intermediate is converted to epoxymexrenone or another compound of formula I using any of the methods described above for method 1.

The advantage of Method 9 is that not only can control the stereochemistry provided by the 5,7-lactone intermediate, but also has the further advantage that a wider range of hydrolysis conditions is possible without the interference of 17-spirolactone.

Like the reactions for other synthetic methods of the present invention, the reactions of Method 9 can be used for the conversion of substrates other than those specifically described above. Thus, for example, the conversion of 3-keto- or 3-ketal-7-cyano steroids to 3-keto- or 3-ketal-5,7-lactone, or 3-keto- or 3-ketal-5, The conversion of 7-lactone to 7α-alkoxycarbonyl can be carried out on compounds substituted by R ⁸ and R ⁹ as defined above at 17-carbon, or more particularly by substituents of the formula:

(Formula XXXIV)

Wherein X, Y ¹ and Y ² are as defined above and C (17) represents 17-carbon. However, an important advantage, in particular in the economics of the method, is the specific reaction method described above for the use of 17-keto substrates followed by the introduction of 17-spirolactone and 7α-alkoxycarbonyl into 3-keto-Δ ^9,11 steroids. By using a specific reaction sequence using

Lactones of formulas D, E and F are novel compounds useful for the preparation of epoxymexrenone and other compounds of formula I and formula IA according to the synthesis of method 9. In these compounds, -AA- and -BB- are preferably -CH ₂ -CH ₂ -and R ³ is hydrogen, lower alkyl or lower alkoxy. Most preferred compounds of formula D are those wherein R ¹⁷ is methoxy.

In a particularly preferred embodiment, the overall method of method 9 proceeds as follows:

Method 10

Method 10 is identical to Method 9 through the formation of the 7-cyano intermediate of Formula C. In the next step of method 10, the 7-cyano steroid is reacted with trialkyl ortho formate in an alkanol solvent, preferably trimethyl ortho formate in methanol, such that the 3-keto group is an enol ether, a 17-keto group Is converted to ketal, thereby protecting both 3-keto and 17-chito groups. The 7-cyano group is then reduced by reaction with 7-formyl, such as a dialkyl aluminum hydride, preferably diisobutyl aluminum hydride, to give a compound of formula 203:

Formula 203

Wherein -AA-, R ³ , and -BB- are as defined in formula XIII, and R ¹⁸ is C ₁ -C ₄ alkyl. Prior protection of the keto group as described above hinders the reduction of the keto group by the dialkyl aluminum hydride. The intermediate of formula 203 is then reacted with diluted aqueous acid to selectively hydrolyze 17-ketal in the presence of excess alcohol (R ¹⁹ OH) to give the intermediate of formula 204:

Wherein R ¹⁹ is selected from lower alkyl (preferably C ₁ to C ₄ ), or the R ¹⁹ group in the 3-position forms a cyclic O, O-oxyalkyleneoxy substituent at the 3-position. Hemiacetal [204] is further protected by treatment with alkanol (R ²⁰ OH) in the presence of non-aqueous acid to give the intermediate of formula 205:

Wherein -AA-, -BB-, R ³ and R ¹⁹ are as defined above and R ²⁰ is C ₁ to C ₄ alkyl. The 17-spirolactone moiety is then introduced following the reaction steps described above for methods 3 and 6, thus proceeding according to the following scheme:

In the above formulas, -AA-, -BB-, R ³ , R ¹⁹ and R ²⁰ are as defined above and R ²⁵ is C ₁ to C ₄ alkyl.

The 3-position is then deprotected by conventional hydrolysis to reintroduce the 3-keto group and the 5,7-hemiacetal to produce additional intermediates corresponding to Formula 209:

Wherein -AA-, -BB- and R ³ are as defined above. The 9,11 epoxide moiety is then introduced according to any of the methods described above for the conversion of the compound of formula II to the compound of formula I. Under the oxidation conditions of the epoxidation reaction, hemiacetal is partially converted to 5,7-lactone, resulting in additional intermediates corresponding to Formula 211:

Wherein -AA-, -BB- and R ³ are as defined above.

All remaining 9,11-epoxy-5,7-hemiacetal intermediate reaction products of the formula 210:

(Wherein -AA-, -BB- and R ³ are as defined above)

Is a compound of formula 211 that is easily oxidized by conventional means. Finally, the intermediate of formula 211 is converted to epoxymexrenone or another compound of formula I using the method described in Method 8 for the conversion of 5,7-lactone to 7α-alkoxycarbonyl compound. Therefore, the method of the overall method 10 proceeds as follows, and it is believed that at least the following steps can be carried out in place without recovery of the intermediate. The overall method of method 10 proceeds as follows:

As in the case of method 9, the reactions described above for method 10 provide important advantages, particularly with regard to the economics of the method; However, the novel reactions of Method 10 also apply more generally to substrates other than those specifically described. For example, the introduction of a 7-formyl group into 3-enol ether steroids, the resulting protection of 7-formyl-Δ-5,6-3,4-enol ether, a valence to 5,7-hemiacetal Degradation, and subsequent deprotection, may be performed on steroids substituted by R ⁸ and R ⁹ as defined above at the 17-position, or more particularly by substituents of the formula:

(Formula XXXIV)

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

Alternative methods of Method 10 include using various intermediates that are each within the range of Equations A203 to A210 as described above. Each of the intermediates of Formulas A203 to A211 are novel compounds useful for the preparation of epoxymexrenone and other compounds of Formula I and Formula IA following the synthesis of Method 10.

In a particularly preferred embodiment, the overall method of method 10 proceeds as follows:

From the several methods exemplified above, it will be appreciated that the reaction steps selected for use in the methods of the present invention provide substantial flexibility in the preparation of epoxymexrenone and related compounds. Important features include, among others: (a) bioconversion of canrenone, androstenedione, or β-sitosterol to 11α- or 9α-hydroxy derivatives (spontaneous conversion of β-sitosterol to 17-keto structure). Conversion is included); (b) introduction of 9,11 double bonds by dehydration of a compound comprising either 11α- or 9α-hydroxy groups, followed by introduction of epoxy groups by oxidation of 9,11 double bonds; (c) adhesion of 7α-alkoxycarbonyl by formation of enamines, hydrolysis of enamines to diketones, and reaction of diketones with alkali metal alkoxides; (d) formation of a 20-spiroxane ring at position 17; (e) formation of 5,7-lactone and esterification of lactone with 7-alkoxycarbonyl; (f) Protection of 3-ketones by conversion to 3-enol ether or 3-ketal during various conversions at different positions (including the formation of 20-spiroxane rings at 17-positions). With some limitations, these four component method elements (b) to (d) can be performed in almost any order. Elements (e) and (f) of the method provide comparable flexibility. These steps provide a route to epoxyxrenone and other compounds of formula I, which are much simpler compared to the method of US Pat. No. 4,559,332. Moreover, these steps provide important advantages in terms of productivity and yield.

In the description of the reaction method as described above, the recovery, isolation and purification of the reaction product can generally be carried out by methods well known to those skilled in the art. Unless stated otherwise, the conditions, solvents, and reagents are conventional ones, are not critical to the consultation, or both. However, certain of the specific processes specifically described above contribute to the excellent overall yield and / or productivity of the various process steps and method and / or the high quality of the intermediates and ultimately 9,11-epoxysteroid products. .

The utility of 20-spiroxane compounds prepared according to the present invention is described in Grob, US Pat. No. 4,559,332.

The 20-spiroxane compounds prepared according to the invention are distinguished by their good biological properties and are therefore valuable pharmaceutically active ingredients. For example, they have strong aldosterone-antagonist action in that they reduce and normalize excessively high sodium retention and potassium secretion induced by aldosterone. They are therefore applied as potassium-saving diuretics, for example for therapeutic purposes important in the treatment of hypertension, heart inadequacy or cirrhosis.

20-spiroxane derivatives having aldosterone-antagonist action are known [eg Fieser and Fieser: Steroids; p 708, Reinhold Publ. Corp., New York, 1959; British Patent Specification No. 1,041,534]. Also known are 17β-hydroxy-21-carboxylic acids and their salts which show similar activity (eg US Pat. No. 3,849,404). However, these types of compounds that have been used for treatment up to now have significant disadvantages in that they always possess certain sex-specific activities which lead to problematic consequences soon or later upon customary long-term treatment. Particularly undesirable are the problematic effects believed to be in the anti-androgenic activity of known anti-aldosterone formulations.

The methods, procedures and compositions of the present invention and the conditions and reagents used therein are further described in the Examples below.

It is a primary object of the present invention to provide an improved process for the preparation of epoxymexrenone, other 20-spiroxanes and other steroids having common structural features. It is a characteristic object of the present invention to provide an improved process for preparing the product of general formula IA and other related compounds in high yield; Providing such a manufacturing method comprising a minimum isolation step; It is to provide such a method that can provide a reasonable capital cost and operate at a reasonable conversion cost.

Accordingly, the present invention provides a series of synthetic methods for epoxymexrenone; Intermediates useful for the preparation of epoxymexrenone; And the synthesis of such novel intermediates.

The novel synthetic methods are described in detail in the description of preferred embodiments. Among the novel intermediates of the present invention are the following.

The compound of formula IV has the structure:

Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group;

R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, halo, hydroxy, lower alkyl, lower alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxy carbonyl, cyano and aryloxy groups;

R ¹ represents an alpha-oriented lower alkoxycarbonyl or hydroxycarbonyl group;

R ² is an 11α-leaving, the separation of which is effective to create a double bond between 9- and 11-carbon atoms;

-BB- is a -CHR ⁶ -CHR ⁷ -group or the following group III in alpha- or beta- orientation:

Formula III

Wherein R ⁶ and R ⁷ are independently hydrogen, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Selected from the group);

R ⁸ and R ⁹ are independently a group consisting of hydrogen, hydroxy, halo, lower alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or together with R ⁶ or R ⁷ , a carbocyclic or heterocyclic type fused to a 5-membered cyclic D ring It constitutes a ring structure.

Compounds of formula IVA correspond to compounds of formula IV wherein R ⁸ and R ⁹ together with the carbon to which they are attached form the following structures:

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

Compounds of formula IVB correspond to compounds of formula IV wherein R ⁸ and R ⁹ together form the structure of formula XXXIII:

The compounds of the formulas IVC, IVD and IVE are each -AA- and -BB- each being -CH ₂ -CH _2- , R ³ is hydrogen, R ¹ is alkoxycarbonyl, preferably methoxycarbonyl Corresponds to the compound of formula IV, IVA, or IVB. Compounds that fall within the scope of formula IV can be prepared by reacting a lower alkylsulfonylating or acylating agent, or halide generating agent, with a corresponding compound falling within the range of formula V.

Compounds of formula V correspond to the following structures:

Wherein -AA-, -BB-, R ¹ , R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula VA correspond to compounds of formula V wherein R ⁸ and R ⁹ together with the carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula VB correspond to compounds of formula IV wherein R ⁸ and R ⁹ together form the structure of formula XXXIII:

(Formula XXXIII)

The compounds of the formulas VC, VD and VE each represent -AA- and -BB- each being -CH ₂ -CH _2- , R ³ is hydrogen, R ¹ is alkoxycarbonyl, preferably methoxycarbonyl Corresponds to the compound of formula V, VA, or VB. Compounds within the scope of formula V can be prepared by reacting alkali metal alkoxides with the corresponding compounds of formula VI.

The compound of formula VI corresponds to the structure:

Formula VI

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula VIA correspond to formula VI in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula VIB correspond to formula VI in which R ⁸ and R ⁹ together form the following formula XXXIII:

(Formula XXXIII)

Compounds of the formulas VIC, VID and VIE correspond to formula VI, VIA, or VIB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds of formula VI, VIA, VIB and VIC are prepared by hydrolyzing the corresponding compounds of formula VII, VIIA, VIIB or VIIC, respectively.

The compound of formula VII corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula VIIA correspond to formula VII wherein R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula VIIB correspond to formula VII wherein R ⁸ and R ⁹ together form formula XXXIII:

(Formula XXXIII)

The compounds of the formulas VIIC, VIID and VIIE correspond to any of formulas VII, VIIA, or VIIB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula VII can be prepared by cyanating compounds within the scope of formula VIII.

Compounds of formula VIII correspond to the following structures:

Wherein, -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV above.

Compounds of formula VIIIA correspond to formula VIII in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula VIIIB correspond to formula VIII wherein R ⁸ and R ⁹ together form the formula XXXIII:

(Formula XXXIII)

Compounds of formulas VIIIC, VIIID and VIIIE correspond to any one of formulas VIII, VIIIA, or VIIIB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula VIII are prepared by oxidizing a substrate comprising a compound of formula XXX, as described below, by fermentation effective to introduce 11-hydroxy groups into the substrate in a-orientation.

Compounds of formula IX correspond to the following structures:

Wherein, -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV and R ¹ is as defined in formula V.

Compounds of formula IXA correspond to formula IX in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula IXB correspond to formula IX in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form formula XXXIII:

(Formula XXXIII)

The compounds of formula IXC, IXD and IXE correspond to any of formulas IX, IXA, or IXB, wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula IX can be prepared by in vivo conversion of the corresponding compound within the scope of formula X.

Compounds of formula XIV correspond to the following structures:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula XIVA correspond to formula XIV in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula XIVB correspond to formula XIV in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIII:

(Formula XXXIII)

The compounds of the formulas XIVC, XIVD and XIVE correspond to any of formulas XIV, XIVA, or XIVB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula XIV can be prepared by hydrolyzing corresponding compounds within the scope of formula XV.

The compound of formula XV corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula XVA correspond to formula XV in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula XVB correspond to formula XV in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the structure of formula XXXIII:

(Formula XXXIII)

Compounds of formulas XVC, XVD and XVE correspond to any of formulas XV, XVA, or XVB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula XV can be prepared by cyanating the corresponding compound within the scope of formula XVI.

The compound of formula XXI corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula XXIA correspond to formula XXI in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula XXIB correspond to formula XXI wherein R ⁸ and R ⁹ together form the structure of formula XXXIII:

(Formula XXXIII)

Compounds of formulas XXIC, XXID and XXIE correspond to any of formulas XXI, XXIA, or XXIB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula XXI can be prepared by hydrolyzing the corresponding compounds within the scope of formula XXII.

The compound of formula XXII corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula XXIIA correspond to formula XXII in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structure:

(Formula XXXIV)

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

Compounds of formula XXIIB correspond to formula XXII wherein R ⁸ and R ⁹ together form the structure of formula XXXIII:

(Formula XXXIII)

Compounds of formulas XXIIC, XXIID and XXIIE correspond to any of formulas XXII, XXIIA, or XXIIB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula XXII can be prepared by cyanating compounds within the scope of formula XXIII.

The compound of formula XXIII corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula XXIIIA correspond to formula XXIII in which R ⁸ and R ⁹ together with the ring carbons attached to them form the following structure:

(Formula XXXIV)

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

Compounds of formula XXIIIB correspond to formula XXIII wherein R ⁸ and R ⁹ together form the structure of formula XXXIII:

(Formula XXXIII)

Compounds of formulas XXIIIC, XXIIID and XXIIIE correspond to any of formulas XXIII, XXIIIA, or XXIIIB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula XXIII can be prepared by oxidizing the compound of formula XXIV as described below.

The compound of formula XXVI corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

The compound of formula XXVIA corresponds to formula XXVI wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula XXVI can be prepared by oxidizing a compound within the scope of formula XXVII.

The compound of formula XXV corresponds to the structure:

Wherein -AA-, -BB-, R ³ , R ⁸ and R ⁹ are as defined in formula IV.

The compound of formula XXVA corresponds to formula XXV wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula XXV can be prepared by cyanating the compound of formula XXVI.

The compound of formula 104 corresponds to the structure:

Wherein -AA-, -BB- and R ³ are as defined in formula IV and R ¹¹ is C ₁ to C ₄ alkyl.

The compound of formula 104A corresponds to formula 104 in which each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of Formula 104 may be prepared by thermal decomposition of the compound of Formula 103.

The compound of formula 103 corresponds to the structure:

Wherein -AA-, -BB-, R ³ and R ¹¹ are as defined in formula 104 and R ¹² is C ₁ to C ₄ alkyl.

The compound of Formula 103A corresponds to Formula 103 wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula 103 can be prepared by reacting the corresponding compound of formula 102 with dialkyl malonate in the presence of a base such as an alkali metal alkoxide.

The compound of formula 102 corresponds to the structure:

Wherein -AA-, -BB-, R ³ and R ¹¹ are as defined in equation 104.

The compound of Formula 102A corresponds to Formula 102 wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula 102 can be prepared by reacting the corresponding compound of formula 101 with a trialkyl sulfonium compound in the presence of a base.

The compound of formula 101 corresponds to the structure:

Wherein -AA-, -BB-, R ³ and R ¹¹ are as defined in equation 104.

The compound of Formula 101A corresponds to Formula 101 wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula 101 can be prepared by reacting 11α-hydroxyandrosten-3,17-dione or another compound of formula XXXVI with trialkyl orthoformate in the presence of an acid.

The compound of formula XL corresponds to the formula

Wherein -E-E- is the following groups:

And

R ²¹ , R ²² and R ²³ are independently selected from hydrogen, alkyl, halo, nitro, and cyano; R ²⁴ is selected from hydrogen and lower alkyl; R ⁸⁰ and R ⁹⁰ are independently selected from keto and substituents which may constitute R ⁸ and R ⁹ (as defined above for Formula IV); -AA-, -BB- and R ³ are as defined in formula IV.

Compounds of formula XLA correspond to formula XL wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

Compounds of formula XLB correspond to formula XLA wherein -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII. The compound of formula XLC corresponds to formula XLB in which -E-E- corresponds to formula XLV. The compound of XLD corresponds to formula XLB wherein -E-E- corresponds to formula XLVII.

Compounds of formula XLE correspond to keto or formula XL together with ring carbon atoms to which R ⁸⁰ and R ⁹⁰ are attached thereto:

(Formula XXXIV)

(Wherein X, Y ¹ , Y ² and C (17) are as defined above);

or

(Formula XXXIII)

The compound of formula XLIE corresponds to formula XL wherein R ⁸⁰ and R ⁹⁰ together form a keto.

Compounds of the formulas XLF, XLG, XLH, XLJ, XLM, and XLN are each represented by compounds of the formulas XL, XLA, XLB, XLC, XLD and XLE, wherein -AA-, -BB- and R ³ are as defined above. Corresponding.

The compound of formula XLI corresponds to the formula:

Wherein -E-E- is the following groups:

Formula XLIII

Formula XLV

(Formula XLVI)

And

Formula XLVII

R ¹⁸ is C ₁ to C ₄ alkyl or R ¹⁸ O- groups together form an O, O-oxyalkylene bridge; R ²¹ , R ²² and R ²³ are independently selected from hydrogen, alkyl, halo, nitro, and cyano; R ²⁴ is selected from hydrogen and lower alkyl; R ⁸⁰ and R ⁹⁰ are independently selected from keto and substituents which may constitute R ⁸ and R ⁹ ; -AA-, -BB- and R ³ are as defined in formula IV.

Compounds of formula XLIA correspond to formula XLI wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

Compounds of formula XLIB correspond to formula XLIA in which -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

Compounds of formula XLIC correspond to keto or formula XLI, together with the ring carbon atoms to which R ⁸⁰ and R ⁹⁰ are attached thereto:

(Formula XXXIV)

(Wherein X, Y ¹ , Y ² and C (17) are as defined above)

Compounds of formula XLID correspond to formula XLI wherein substituent XXXIV corresponds to formula XXXIII:

(Formula XXXIII)

Compound of formula XLIE corresponds to formula XLI wherein R ⁸⁰ and R ⁹⁰ together form a keto.

Compounds of the formulas XLIF, XLIG, XLIH, XLIJ, XLIM, and XLIN are each represented by compounds of the formulas XLI, XLIA, XLIB, XLIC, XLID and XLIE, wherein -AA-, -BB- and R ³ are as defined above. Corresponding. Compounds within the scope of formula XLI are prepared by hydrolyzing the corresponding compounds of formula XL as defined below.

The compound of formula XLII corresponds to the formula:

Wherein -E-E- is the following groups:

Formula XLIII

Formula XLIV

Formula XLV

(Formula XLVI)

And

Formula XLVII

R ²¹ , R ²² and R ²³ are independently selected from hydrogen, alkyl, halo, nitro, and cyano; R ²⁴ is selected from hydrogen and lower alkyl; R ⁸⁰ and R ⁹⁰ are independently selected from keto and substituents which may constitute R ⁸ and R ⁹ ; -AA-, -BB- and R ³ are as defined in formula IV.

Compounds of formula XLIIA correspond to formula XLII wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

Compounds of formula XLIIB correspond to formula XLIIA in which -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

Compounds of formula XLIIC correspond to keto or formula XLII together with the ring carbons to which R ⁸⁰ and R ⁹⁰ are attached thereto:

(Formula XXXIV)

(Wherein X, Y ¹ , Y ² and C (17) are as defined above)

Compounds of formula XLIID correspond to formula XLII wherein substituent XXXIV corresponds to formula XXXIII:

(Formula XXXIII)

The compound of formula XLIIE corresponds to formula XLII, wherein R ⁸⁰ and R ⁹⁰ together form a keto. The compounds of the formulas XLIIF, XLIIG, XLIIH, XLIIJ, XLIIM, and XLIIN are each represented by the formulas XLII, XLIIA, XLIIB, XLIIC, XLIID and XLIIE, wherein -AA- and -BB- are -CH ₂ -CH ₂ and R ³ is hydrogen. Corresponding. Compounds within the scope of formula XLII are prepared by deprotecting the corresponding compound of formula XLI.

Compounds of formula XLIX correspond to the following formulas:

Wherein -EE- is as defined in formula XL and -AA-, -BB-, R ¹ , R ³ , R ⁸ and R ⁹ are as defined in formula IV.

Compounds of formula XLIXA correspond to formula XLIX in which R ⁸ and R ⁹ together with the ring carbon to which they are attached form the following structural formula:

(Formula XXXIV)

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

Compounds of formula XLIXB correspond to formula XLIX in which R ⁸ and R ⁹ together form the structure XXXIII:

(Formula XXXIII)

Compounds of formulas XLIXC, XLIXD, XLIXE are each of formulas XLIX, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ , R ³ is hydrogen and R ¹ is alkoxycarbonyl, preferably methoxycarbonyl; It corresponds to either XLIXA or XLIXB. Compounds within the scope of formula XLIX may be prepared by reacting an alcoholic or aqueous solvent with the corresponding compound of formula VI in the presence of a suitable base.

The compound of formula A203 corresponds to the following formula:

Wherein -E-E- is the following groups:

Formula XLIII

Formula XLV

(Formula XLVI)

And

Formula XLVII

R ¹⁸ is selected from C ₁ to C ₄ alkyl; R ²¹ , R ²² and R ²³ are independently selected from hydrogen, alkyl, halo, nitro, and cyano; R ²⁴ is selected from hydrogen and lower alkyl; -AA-, -BB- and R ³ are as defined in formula IV.

Compounds of formula A203A correspond to formula A203 wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

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

Compounds of formulas A203C, A203D, and A203E correspond to formulas A203, A203A, and A203B, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula A203 are prepared by reducing the compound of formula A202 as described below.

Compound of Formula A204 corresponds to the following structural formula:

Wherein R ¹⁹ is C ₁ to C ₄ alkyl and -EE-, -AA-, -BB- and R ³ are as defined in formula 203.

Compounds of formula A204A correspond to formula A204 wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

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

Compounds of formulas A204C, A204D, and A204E correspond to formulas A204, A204A, and A204B, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula A204 are prepared by hydrolyzing the corresponding compounds of formula A203.

The compound of formula A205 corresponds to the following structure:

Wherein R ²⁰ is C ₁ to C ₄ alkyl and -EE-, R ¹⁹ , -AA-, -BB- and R ³ are as defined in formula 204.

Compounds of formula A205A correspond to formula A205 wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

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

Compounds of formulas A205C, A205D, and A205E correspond to formulas A205, A205A, and A205B, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula A205 can be prepared by reacting the corresponding compounds of formula A204 with alkanols and acids.

The compound of formula A206 corresponds to the following structural formula:

Wherein R ¹⁹ , R ²⁰ , -EE-, -AA-, -BB- and R ³ are as defined in equation 205.

Compounds of formula A206A correspond to formula A206 wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

Compounds of formula A206B correspond to formula A206A in which -E-E- corresponds to formula XLIII, XLIV, XLV or XLVII.

Compounds of formulas A206C, A206D, and A206E correspond to formulas A206, A206A, and A206B, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the scope of formula A206 can be prepared by reacting corresponding compounds within the scope of formula A205 with trialkyl sulfonium halides.

The compound of formula A207 corresponds to the following structural formula:

Wherein R ²⁵ is C ₁ to C ₄ alkyl and -EE-, R ¹⁹ , R ²⁰ , -AA-, -BB- and R ³ are as defined in formula A205.

Compounds of formula A207A correspond to formula A207 wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, halogen and lower alkyl.

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

Compounds of formulas A207C, A207D, and A207E correspond to formulas A207, A207A, and A207B, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds of formula A207 can be prepared by reacting compounds of formula A206 with dialkyl malonate.

The compound of formula A208 corresponds to the following structure:

Wherein, -EE-, R ⁸⁰ and R ⁹⁰ are as defined in formula XLII; -AA-, -BB- and R ³ are as defined in equation 104; R ¹⁹ , R ²⁰ , -AA-, -BB- and R ³ are as defined in equation 205.

Compounds of formula A208A correspond to formula A208 wherein R ²¹ and R ²² are independently selected from hydrogen, halogen and lower alkyl.

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

Compounds of formula A208C correspond to keto or formula A208 in which R ⁸⁰ and R ⁹⁰ together with the ring carbons attached thereto constitute a formula:

(Formula XXXIV)

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

Compounds of formula A208D correspond to formula A208C wherein substituent XXXIV corresponds to formula XXXIII:

(Formula XXXIII)

Compounds of formulas A208E, A208F, A208G, A208H and A208J correspond to formulas A208, A208A, A208B, A208C and A208D, wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen do. Compounds within the scope of formula A208 can be prepared by thermal decomposition of the corresponding compounds of formula A207.

The compound of formula A209 corresponds to the following structural formula:

Wherein R ⁸⁰ and R ⁹⁰ are as defined in formula XLI, and -EE- and -AA-, -BB- and R ³ are as defined in formula 205.

Compounds of formula A209A correspond to formula A209 wherein R ²¹ and R ²² are independently selected from hydrogen, halogen and lower alkyl.

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

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

Compounds of formula A209D correspond to keto or formula A208 in which R ⁸⁰ and R ⁹⁰ together with the ring carbons attached thereto constitute a formula:

(Formula XXXIV)

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

Compounds of formula A209E correspond to formula 209D wherein substituent XXXIV corresponds to formula XXXIII:

(Formula XXXIII)

Compounds of formulas A209F, A209G, A209H, A209J, A209L and A209M are each of formulas A209, A209A, A209B, A209C and A209D, wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen And A209E. Compounds within the scope of formula A209 can be prepared by hydrolyzing the corresponding compounds of formula A208.

The compound of formula A210 corresponds to the following structure:

Wherein R ⁸⁰ and R ⁹⁰ are as defined in formula XLI and the substituents -AA-, -BB- and R ³ are as defined in formula IV.

Compounds of formula A210A correspond to keto or formula A210, in which R ⁸⁰ and R ⁹⁰ together with the ring carbons attached thereto constitute the following formula:

(Formula XXXIV)

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

Compounds of formula A210B correspond to formula A210A wherein substituent XXXIV corresponds to formula XXXIII:

(Formula XXXIII)

Compound of formula A210C corresponds to formula A210A wherein R ⁸⁰ and R ⁹⁰ together form a keto.

Compounds of formulas A210D, A210E, A210F and A210G correspond to formulas A210, A210A, A210B and A210C, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen. Compounds within the range of Equation 210 are -EE- It can be prepared by epoxidizing the compound of formula 209.

The compound of formula A211 corresponds to the formula:

Wherein -AA-, -BB- and R ³ are as defined above.

Compounds of formula A211A correspond to formula A211 wherein R and R together constitute a keto or the following formula:

(Formula XXXIV)

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

Compounds of formula A211B correspond to formula A211A wherein substituent XXXIV corresponds to formula XXXIII:

(Formula XXXIII)

Compounds of formula A211C correspond to formula A210A, wherein R ⁸⁰ and R ⁹⁰ together form a keto.

Compounds of formulas A211D, A211E, A211F, and A211G correspond to formulas A211, A211A, A211B and A211C, wherein each of -AA- and -BB- is -CH ₂ -CH ₂ -and R ³ is hydrogen. Compounds within the scope of formula A211 can be prepared by oxidizing the corresponding compound of formula A210; Or in the process of epoxidizing the corresponding compound of formula A209 wherein -EE- is C = CH-. The compounds of formula A211 can be converted to the compounds of formula I in the manner described below.

 Compounds of formula L correspond to the following structural formula:

Wherein R ¹¹ is C ₁ to C ₄ alkyl and -AA-, -BB-, R ¹ , R ² , R ³ , R ⁸ and R ⁹ are as defined above.

Compounds of formula LA correspond to formula L in which R ⁸ and R ⁹ together with the carbon atom to which they are attached form:

(Formula XXXIV)

Wherein X, Y ¹ and Y ² are as defined above.

Compounds of formula LB correspond to formula L wherein R ⁸ and R ⁹ correspond to formula XXXIII:

(Formula XXXIII)

The compounds of the formulas LC, LD, LE correspond to formulas L, LA and LB, wherein each of —AA— and —BB— is —CH ₂ —CH ₂ — and R ³ is hydrogen.

Based on the description of the specific reaction methods described below, it will be evident that these compounds are very much utilized for certain reaction methods. The compounds of the present invention are useful as intermediates for epoxymexrenone and other steroids.

Other objects and features will be in part apparent and in part pointed out hereinafter.

Example 1

The slope was prepared with the growth medium shown in Table 1.

Y P D A (badge for slopes and plates) Yeast extract 20 g peptone 20 g Glucose 20 g Agar 20 g With an appropriate amount of distilled water to 1000ml and the pH is 6.7 and the circle .- _{-H 3 PO 4 10% w /} v is adjusted to pH 5 with Dispense-to-Slope: 7.5 ml-flat for 180 x 18 mm tubes (10 cm in diameter) in 200 x 20 mm tubes at 25 ml-120 ° C.

To produce first generation cultures, colonies of Aspergillus ochraceus were suspended in distilled water (2 ml) in vitro; 0.15 ml of this suspension was inoculated on each of the four slopes prepared as described above. After culturing the slope at 25 ° C. for 7 days, the surface of the culture medium was the appearance of white downy mycelium. The back side was colored orange at the bottom and yellow-orange at the top.

The first generation slope cultures were suspended in a sterile solution (4 ml) containing Tween 80 nonionic surfactant (3% by weight), and the second generation slope prepared from the growth medium shown in Table 2 using 0.15 ml of this suspension. Inoculated.

(For second generation and normal slope) Malt Extract 20 g peptone 1 g Glucose 20 g Agar 20 g Make an appropriate amount of distilled water to 1,000 ml.-Original pH is 5.3-Dispense to 7.5 ml in tubes (180 x 18 mm)-Sterilize for 20 minutes at 120 ° C.

Second generation slopes were incubated at 25 ° C. for 10 days, producing large amounts of gold spores; The back side is colored brown orange.

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

Protection Skim milk 10 g Distilled water 100 ml Skim milk is added to a 250 ml flask containing 100 ml distilled water at 50 ° C. Sterilize at 120 ° C for 15 minutes. Cool at 33 ° C and use in the day.

Cultures from five of the second generation slopes were suspended in a protective solution (15 ml) in a 100 ml flask. The suspension was dispensed 0.5 ml each in 100 x 10 mm tubes for lyophilization. This was pre-frozen for 20 minutes at -70 ° C to -80 ° C in acetone / dry ice bath and immediately transferred to a drying chamber previously cooled to -40 ° C to -50 ° C. The pre-frozen portions were lyophilized at a residual pressure of 50 μHg and ≦ 30 ° C. After lyophilization was completed, two to three granules of sterile silica gel were added to each tube along with a humidity indicator and flame seal. In order to obtain suitable parental culture slopes for industrial scale fermentation, one fraction of the lyophilized cultures prepared in the above manner was suspended in distilled water (1 ml) and the suspension was used with 0.15 ml each to have the composition shown in Table 2. Inoculated on slopes to provide growth medium. The woolen slopes were incubated at 25 ° C. for 7 days. After incubation, the cultures grown at the slope were stored at 4 ° C.

To prepare conventional slope cultures, the cultures from the parental slopes were suspended in sterile solution (4 ml) containing Tween 80 (3% by weight), and the resulting suspension was covered with growth media as described in Table 2. 0.15 ml was distributed to the fields. Conventional slope cultures can be used to inoculate primary inoculation flasks for research or industrial fermentation.

To prepare the first inoculated flask culture, the culture from the usual slope prepared as described above was removed and suspended in a solution containing Tween 80 (3% by weight). Each of the resulting suspensions was injected into a 500 ml baffle flask containing growth medium having the composition shown in Table 4.

(For primary and transformed flask cultures and round bottom flasks) Glucose 20 g peptone 20 g Yeast self-lysate 20 g With an appropriate amount of distilled water-adjusted to pH 5.8 with -20% NaOH-100 ml in a 500 ml baffle flask-500 ml each with a 2000 ml round bottom flask-sterilized for 20 minutes at 120 ° C-after sterilization Set the pH to about 5.7.

  The inoculating flasks were incubated for 24 hours at 28 ° C. on a rotary shaker (200 rpm, 5 cm displacement), thereby producing culture in the form of mycelium similar to pellets having a diameter of 3 to 4 mm. Microscopic observation revealed that the species culture was concentrated mycelium and was pure culture with large mycelia that were well twisted. The pH of the suspension was 5.4 to 5.6. PMV was 5-8% as determined by centrifugation (3000 rpm x 5 min).

Transformed flask cultures were prepared by inoculating biomass (1 ml) from a species culture flask into a growth medium (100 ml) having the composition shown in Table 4 in a second 500 ml shaker flask. The resulting mixture was incubated at 28 ° C. for 18 hours on a rotary shaker (200 rpm, 5 cm displacement). The cultures were examined and found to contain mycelium similar to pellets having a diameter of 3-4 mm. By microscopic examination, the cultures were determined to be pure cultures with neoplastic cells in the cytoplasm and the old cells with a focused mycelial and island-like growth with little fear. The pH of the culture suspension was between 5 and 5.2 and PMV was between 10% and 15% as determined by centrifugation. Therefore, the culture was considered to be suitable for transformation from canrenone to 11α-canylene hydroxide.

Canrenone (1 g) was ground to about 5 mu and suspended in sterile water (20 ml). 40% (w / v) sterile glucose solution; Sterile solution of 16% (w / v) autolysed yeast; And sterile antibiotic solutions; All of these were added to the suspension at the rates indicated for 0 hours of the reaction time in Table 5. The antibiotic solution was prepared by dissolving kanamycin sulfate (40 mg), tetracycline hydrochloride (40 mg) and cephalexin (200 mg) in water (100 ml). Steroid suspensions, glucose solutions and autolysed yeast solutions were gradually added to the cultures contained in shake flasks.

Instruction for the addition of steroids and solutions (additives and antibiotics) during the bioconversion of canrenone in shake flasks Reaction time (hours). Steroid suspension.ml approx.mg. Glucose solution.ml Yeast autolysate solution. ml Antibiotic solution.ml 0 One 50 One 0.5 One 8 2 100 2 One 24 2 100 One 0.5 One 32 5 250 2 One 48 2 100 One 0.5 One 56 5 250 2 One 72 3 150 One 0.5 One 90

As the reaction progressed, the reaction mixture was analyzed periodically to determine the glucose content and the conversion to 11α-canrenone hydroxide by thin layer chromatography. During the reaction additional canrenone substrates and nutrients were added to the fermentation reaction mixture at a rate adjusted to keep the glucose content in the range of about 0.1% by weight. The addition methods for steroid suspensions, glucose solutions, autolysed yeast solutions and antibiotic solutions are shown in Table 5. Transformation was continued for 25 hours at 25 ° C. on a rotary shaker (200 rpm and 5 cm displacement). During fermentation, the pH ranged between 4.5 and 6. When the PMV rose to or above 60%, the 10 ml portion of the broth was removed and replaced with 10 ml of distilled water. Disappearance of canrenone and the appearance of 11α-canned hydroxide were tracked during the reaction by sampling the juice at intervals of 4, 7, 23, 31, 47, 55, 71, 80 and 96 hours after the start of the fermentation cycle. Analyzed by TLC. The progress of the reaction determined from this sample is shown in Table 6.

Time-lapse of Canrenon Bioconversion in Shake Flasks Time (hours) Conversion ratio of Kanenon R _f . 11α-cananyl hydroxide R _f . = 0.81 R _f . = 0.29 0 100 0.0 4 50 50 7 20 80 23 20 80 31 30 70 47 20 80 55 30 70 71 25 75 80 15 85 96 To 10 ～ 90

Example 2

Main inoculation flask cultures were prepared in the manner described in Example 1. A nutritive mixture was prepared having the composition shown in Table 7.

For transformed culture in 10 l glass fermenter Yang g / l Glucose 80 g 20 peptone 80 g 20 Yeast Soluble 80 g 20 Antifoam SAG 471 0.5 g 4 L of deionized water in an appropriate amount-sterilize the empty fermenter for 30 minutes at 130 ° C-fill it with 3 L of deionized water, heat at 40 ° C-add with stirring the components of the medium-stir for 15 minutes, volume to 3.9 l The original pH is adjusted to 5.8 with 5.1-20 w / v% NaOH, sterilized at 120 ° C. for 20 minutes, and the pH after sterilization is 5.5 to 5.7. The initial amount of this nutrient mixture (4 L) was injected into a 10 L geometric volume transformed fermenter. The fermenter was cylindrical in shape with a height to diameter ratio of 2.58. It is equipped with a 400 rpm turbine stirrer with two No. 2 disc wheels with six blades each. The outer diameter of the impeller is 80 mm, each blade is 25 mm and 30 mm high in the radial dimension, the wheel on the top is 280 mm below the top of the vessel, and the wheel on the bottom is 365 mm below the top of the vessel. The septum is 210 mm high and 25 mm radially inward from the inner vertical wall of the vessel.

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Species cultures (40 ml) were mixed with the amount of nutrients in the fermenter and incubated at 28 ° C. for 22 hours and the transgenic medium was settled at aeration rate of 0.5 l / l per minute at a pressure of 0.5 kg / cm ² . At 22 hours, the culture medium had a PMV of 20-25% and a pH of 5 to 5.2. A suspension comprising canrenone (80 g) in sterile water (400 ml) was prepared and a 10 ml portion was added to the mixture in the transfection fermenter. At the same time, 40% (w / v) sterile glucose solution, 16% (w / v) autolysed yeast sterile solution and sterile antibiotic solution were added at the rate indicated in the reaction time of 0 hours in Table 8. Antibiotic solutions were prepared in the manner described in Example 1.

10 l Instructions for the addition of steroids and solutions (additives and antibiotic solutions) during the bioconversion of canrenone in glass fermenters. Response time (hours) Steroid suspension approxml gr Glucose solution ml Yeast autolysate ml Antibiotic solution ml 0 10 4 25 12.5 40 4 25 12.5 8 10 4 25 12.5 12 25 12.5 16 10 4 25 12.5 20 25 12.5 24 10 4 25 12.5 40 28 10 4 25 12.5 32 12.5 5 25 12.5 36 12.5 5 25 12.5 40 12.5 5 25 12.5 44 12.5 5 25 12.5 48 12.5 5 25 12.5 40 52 12.5 5 25 12.5 56 12.5 5 25 12.5 60 12.5 5 25 12.5 64 12.5 5 25 12.5 68 12.5 5 25 12.5 72 12.5 5 25 12.5 40 76 12.5 5 25 12.5 80 84 88

As the reaction progressed, the reaction mixture was analyzed periodically to determine the glucose content, and the conversion to 11α-canrenone hydroxide was determined by thin layer chromatography. Based on the TLC analysis of the reaction broth samples described below, additional canrenones were added to the reaction mixture as the canrenone substrate was consumed. Glucose levels were also tracked and when the glucose concentration dropped to or below about 0.05% by weight, a supplemental glucose solution was added to raise the concentration to 0.25% by weight. Nutrients and antibiotics were also added discontinuously during the reaction cycle. The addition methods for steroid suspensions, glucose solutions, self-dissolved yeast solutions and antibiotic solutions are shown in Table 8. The transformation reaction continued for 90 hours at an aeration rate of 0.5 volume air (vvm) per liquid volume per minute at an initial positive pressure of 0.3 kg / cm ² . The temperature was held at 28 ° C. until the PVM reached 45%, then lowered to 26 ° C. at this temperature until the PVM reached 45-60%, and then adjusted to 24 ° C. The initial stirring speed was 400 rpm and after 40 hours it increased to 700 rpm. The pH was maintained between 4.7 and 5.3 by addition of 2M orthophosphoric acid or 2M NaOH as indicated. When foaming occurred, the foam was controlled by adding a few drops of the antifoam SAG 471. The loss of canrenone and the appearance of 11α-canned hydroxide were tracked at intervals of 4 hours during the reaction by TLC analysis of the juicy samples.

After the addition of all canrenones, the reaction was terminated when TLC analysis showed that the concentration of the canrenone substrate for 11α-hydroxynrenone dropped to about 5%.

At the end of the reaction cycle, the fermented broth was filtered with a cotton cloth for separation of mycelium from the liquid broth. Mycelial fractions were resuspended in ethyl acetate using about 65 volumes (5.2 liters) per gram of canrenone consumed during the reaction. The suspension of the mycelium in ethyl acetate was redissolved for 1 hour with stirring, cooled to about 20 ° C. and filtered over Buchner. The mycelium Kate was washed sequentially with ethyl acetate (5 volumes per gram of canlenone consumption; 0.4 L) and deionized water (500 ml) to replace the ethyl acetate extract from the cake. Discarded the filter cake. Abundant extracts, solvent washes and water washes were collected in a separator and left for 2 hours for phase separation.

The aqueous phase was then discarded and the organic phase concentrated in vacuo to 350 ml residual volume. The bottom was cooled to 15 ° C. and maintained for about 1 hour with stirring. The resulting suspension was filtered to remove crystalline material and the filter cake was washed with ethyl acetate (40 ml). After drying, the yield of 11α-kanenone hydroxide was determined to be 60 g.

Example 3

Spore suspensions were prepared from conventional slopes in the manner described in Example 1. In a 2000 ml baffle round bottom flask (3 diaphragms, 50 mm x 30 mm each), 0.5 ml of spore suspension was injected into a nutrient solution (500 ml) having the composition shown in Table 4. The resulting mixture was incubated in a flask at 25 ° C. for 24 hours on a separate shaker (120 strokes per minute; 5 cm displacement), whereby the microorganisms observed to be seen as pure cultures with well-twisted mycelium were removed. Created. The pH of the culture was between about 5.3 and 5.5, and PMV (determined by centrifugation at 3000 rpm for 5 minutes) was 8-10%.

Using the cultures thus prepared, species cultures were prepared in a vertical cylindrical stainless steel fermenter with a 160 L geometric volume and a ratio of height to height 2.31 (height = 985 mm; diameter = 425 mm). The fermentor has a disc turbine-shaped stirrer with two wheels, each wheel having an outer diameter of 240 mm and six blades, each blade having an 80 mm radial dimension and a height of 50 mm. The upper wheel is located at a depth of 780 mm and then 995 mm from the top of the fermenter. Vertical baffles with a height of 890 mm extend 40 mm inward from the vertical inner wall of the fermenter. The stirrer was operated at 170 rpm. The nutrient mixture (100L) having the composition shown in Table 9 was put into the fermenter, and then a portion of the priinokulum (1L) having a pH of 5.7 prepared as described above was added.

About 160 liters of growth culture for 160 L fermentation tank is needed for inoculation production fermentation tank. amount g / L Glucose 2 Kg 20 peptone 2 Kg 20 Yeast Soluble 2 Kg 20 Antifoam SAG 471 0.010 Kg a very small amount 100 L of deionized water in an appropriate amount.-Sterilize the empty fermenter for 1 hour at 130 DEG C.-Charge this with 6 L of deionized water; Stir the heat-medium components at 40 ° C, add 15 minutes, stir to a volume of 95L. After sterilization and sterilization at -121 ° C for 30 minutes, the pH is 5.7-sterilized deionized water and 100L

The inoculated mixture was incubated for 22 hours at an aeration rate of 0.5 L / L-min at an initial pressure of 0.5 Kg / cm ² . The temperature was adjusted to 28 ° C. until PMV reached 25% and then agitated to 25 ° C. The pH was adjusted in the range of 5.1 to 5.3. Growth of mycelial volume is shown in Table 10 depending on dissolved oxygen profile and pH of the species culture.

Time course of mycelial growth in seed culture fermenters Fermentation period (hours) pH Aggregated Mycelium Volume (pmv)% (3000 rpms 5 min) Dissolved oxygen% 0 5.7 ± 0.1 100 4 5.7 ± 0.1 100 8 5.7 ± 0.1 12 ± 3 85 ± 5 12 5.7 ± 0.1 15 ± 3 72 ± 5 16 5.5 ± 0.1 25 ± 5 40 ± 5 20 5.4 ± 0.1 30 ± 5 35 ± 5 22 5.3 ± 0.1 33 ± 5 30 ± 5 24 5.2 ± 0.1 35 ± 5 25 ± 5 Using the inoculated culture thus produced, the transformation fermentation operation was performed in a vertical cylindrical stainless steel fermenter having a geometric volume of 1.02 m in diameter, 1.5 m in height and 1.4 m ³ . The fermentor has a turbine stirrer with two impellers, one located at 867 cm below the top of the reactor and the other at 1435 cm from the top. Each wheel has six blades, each with a radial dimension of 95 cm and a height of 75 cm. A vertical baffle 1440 cm high extends radially inwards 100 cm from the vertical inner wall of the reactor. A nutrient mixture was prepared having the composition set shown in Table 11.

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For biotransformation culture in 1000L fermenter amount g / L Glucose 16 Kg 23 peptone 16 Kg 23 Yeast Soluble 16 Kg 23 Antifoam SAG 471 0.080 Kg a very small amount 700 L of deionized water in an appropriate amount. Sterilize the empty fermenter for 1 hour at 130 ° C.—fill with 600 L deionized water; Stir the ingredients of the heat-medium at 40 ° C, stir for 15 minutes, and add a volume of 650 L. After sterilization and sterilization at 121 ° C for 30 minutes, the pH is adjusted to 700 L with 5.7-sterilized deionized water.

The initial amount (700 L) of the nutrient mixture (pH = 5.7) was placed in a fermenter, followed by the inoculum of this example (7 L) prepared as described above.

The nutrient mixture containing the inoculum was incubated for 24 hours at an aeration rate of 0.5 L / L-min at an initial pressure of 0.5 Kg / cm ² . The temperature was adjusted to 28 ° C. and the stirring speed was 110 rpm. Mycelial volume growth is shown in Table 12, depending on the dissolved oxygen profile and pH of the species culture reaction.

Time course of mycelial growth in fermenters of transgenic cultures Fermentation period (hours) pH Aggregated Mycelium Volume (pmv)% (3000 rpm x 5 minutes) Dissolved oxygen% 0 5.6 ± 0.2 100 4 5.5 ± 0.2 100 8 5.5 ± 0.2 12 ± 3 95 ± 5 12 15 ± 3 90 ± 5 16 5.4 ± 0.1 20 ± 5 75 ± 5 20 5.3 ± 0.1 25 ± 5 60 ± 5 22 5.2 ± 0.1 30 ± 5 40 ± 5 At the end of incubation, mycelium pellets were observed, but these pellets were generally small and relatively loosely packed together. Scattered mycelium was suspended in gravy. Final pH was 5.1 to 5.3.

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To this transgenic culture, a suspension of canrenone (1.250 Kg; finely divided into 5 µ) in sterile water (5 L) was added. Sterile addition solution and antibiotic solution were added at the ratio indicated in reaction time 0 shown in Table 14. The composition of the addition liquid is shown in Table 13.

Additives (for transgenic cultures) amount Dextrose 40 Kg Yeast Soluble 8 Kg Antifoam SAG 471 0.010 Kg 100 L of deionized water in an appropriate amount. Sterilize the 150 L empty fermenter for 1 hour at 130 ° C.—fill it with 70 L deionized water; Stir the ingredients of the heat-adding liquid at 40 ° C., stir for 30 minutes, and make up to a volume of 95 L.-The original pH is 4.9-120 ° C. for 20 minutes sterilization-The pH after sterilization is about 5

Bioconversion was performed at a pH in the range of 4.7 to 5.3 controlled by an initial pressure of 0.5 Kg / cm ² , aeration rate of 0.5 L / L-min and addition of 7.5 M NaOH or 4 MH ₃ PO ₄ as needed. The stirring speed was initially 100 rpm, 165 rpm at 40 hours and 250 rpm at 64 hours. The initial temperature was 28 ° C., lowered to 26 ° C. when PMV reached 45%, and lowered to 24 ° C. when PMV rose to 60%. Aesthetic SAG 471 was added as needed to control bubble formation. During the fermentation, glucose levels were followed at 4 hour intervals, and whenever the glucose concentration dropped below 1 gpl, an increase in sterile additive (10 L) was added to the batch. In addition, the disappearance of canrenone and the appearance of 11α-canylene hydroxide were followed by HPLC during the reaction. When at least 90% of the initial canrenone amount was converted to 11α-hydroxylated canrenone, an increase of 1.250 Kg canrenone was added. When 90% of this increased canrenon was converted, it injected another 1.250 Kg. Using the same criteria, a further increase (1.250 Kg at a time) was added until the entire reaction amount (20 Kg) was injected. After the entire amount of canrenone was put into the reactor, the reaction was terminated when the concentration of unreacted canrenone was 5% relative to the amount of 11α-canned hydroxide produced. Table 14 shows the method for the addition of canrenone, sterile additive and antibiotic solution.

Addition of Steroids and Solutions (Additives and Antibiotics) in the Bioconversion of Canrenone in Fermentation Tanks Response time (hours) Kg cumulative Kg in the Kanrenon suspension Sterilization Additives (L) Antibiotic solution (L) Volume (L) approx 0 1.250 1.25 10 8 700 4 10 8 1.250 2.5 10 12 10 16 1.250 10 20 10 24 1.250 5 10 8 800 28 1.250 10 32 1.250 10 36 1.250 10 40 1.250 10 44 1.250 10 48 1.250 12.5 10 8 900 52 1.250 10 56 1.250 10 60 1.250 10 64 1.250 10 68 1.250 10 72 1.250 20 10 8 1050 76 0 80 84 88 92 Sum

When the bioconversion was complete, the broth and mycelium were separated by centrifugation with a basket centrifuge. By HPLC it was determined that the filtrate contained only 2% of the total amount of 11a-canrenone hydroxide in the harvest broth and thus was discarded. The mycelium was suspended in ethyl acetate (1000 L) in a 2 m ³ extraction tank. The suspension was heated for 1 hour under stirred and ethyl acetate redissolved conditions, then cooled and centrifuged in a basket centrifuge. The mycelium cake was washed with ethyl acetate (200 L) and then released. Steroid-rich solvent extracts were allowed to stand for 1 hour for separation of the aqueous phase. The aqueous phase was extracted with an additional amount of ethyl acetate solvent (200 L) and then discarded. The mixed solvent phases were sorted by centrifugation, placed in a concentrator (500 L geometric volume) and concentrated under vacuum to a 100 L residual volume. During the evaporation, the initial amount for the concentrator of the mixed extract and wash was 100 L and this volume was kept constant by periodic or continuous addition of the mixed solution as the solvent evaporated. After the evaporation step was completed, the bottom was cooled to 20 ° C., stirred for 2 h and filtered on a Buchner filter. The concentrator jar was washed with ethyl acetate (20 L) and this wash was used to wash the cake on the filter. The product was dried under vacuum at 50 ° C. for 16 hours. The yield of 11α-kanenone hydroxide was 14 Kg.

Example 4

Dried and frozen spores of Aspergillus ochraceus NRRL 405 were suspended in corn steep liquor growth medium having the composition shown in Table 15.

  Corn steep liquor medium (growth medium for primary species culture) Corn Steep Liquid 30 g Yeast extract 15 g Monobasic Ammonium Phosphate 3 g Add appropriate amount of distilled water to glucose (post-sterilization) to make 1000 ml, adjust the original pH to 4.6, adjust pH to 6.5 with 20% NaOH, and distribute 50 ml to a 250 ml Elenmeyer flask and sterilize at 121 ° C for 20 minutes. 30 g

The resulting suspension was used as inoculum for propagation of spores on agar plates. Ten agar plates were prepared and each contained a solid glucose / yeast extract / phosphate / agar growth medium having the composition shown in Table 16.

GYPA (glucose / yeast extract / phosphate / agar for flat plate) Glucose (amount after sterilization) 10 g Yeast extract 2.5 g K ₂ HPO ₄ 3 g Make 1000 ml of agar distilled water. Adjust pH to 6.5 and sterilize at 121 ℃ for 30 minutes. 20 g

0.2 ml of the suspension were transferred to the surface of each plate. Plates were incubated at 25 ° C. for 10 days, after which spores from all plates were collected and transferred to sterile cryoprotective medium having the composition shown in Table 17.

 GYP / glycerol (glucose / yeast extract / phosphate / glycerol medium for stock vials) Glucose (amount after sterilization) 10 g Yeast extract 2.5 g K ₂ HPO ₄ 3 g Make 1000ml of appropriate amount of distilled water and sterilize at 121 ℃ for 30 minutes. 20 g

The resulting suspension was transferred 1 ml for each vial and divided into 20 vials. This vial constitutes a master cell vane that can be relied upon to produce an operating cell vane for use in the generation of an inoculum for the bioconversion of canlenone's 11α-canylene hydroxide. The vial containing the master cell vank was stored in the vapor phase of the liquid nitrogen cooler at -130 ° C.

To begin production of working cell vanes, spores from one master cell vane vial were resuspended in sterile growth medium (1 ml) having the composition shown in Table 15. The suspension was divided into ten 0.2 ml fractions and each fraction was used to inoculate agar plates containing solid growth medium having the composition shown in Table 16. This plate was incubated at 25 ° C. for 10 days. On day 3 of incubation, the lower part of the growth medium was brown-orange. At the end of the incubation was the mass production of gold spores. Spores from each plate were collected by the method described herein for the production of master cell vanes. A total of 100 vials were prepared, each containing 1 mg of suspension. These vials constitute an operating cell vane. The operating cell vane vials were also stored by preserving in the vapor phase of the liquid nitrogen cooler at -130 ° C.

Growth medium (50 ml) having the composition shown in Table 15 was charged to a 250 ml Ellenmeyer flask. A fraction of the working cell suspension (0.5 ml) was placed in a flask and mixed with the growth medium. The inoculated mixture was incubated at 25 ° C. for 24 hours to produce primary species cultures having a percent aggregated mycelium volume of approximately 45%. By visual inspection, the cultures were found to contain pellet-like mycelium with a diameter of 1 to 2 mm and, by microscopic observation, appeared as pure culture.

The growth of the secondary species culture was initiated by injecting a growth medium having the composition shown in Table 15 into a 2.8 L Perenbach flask and inoculating a portion (10 ml) of the primary species culture of this example in a medium, the preparation of this. Has been described above. The inoculated mixture was incubated for 24 hours at 25 ° C. on a rotary shaker (200 rpm, 5 cm displacement). At the end of the incubation, the cultures showed the same properties as described above for the primary species cultures and were suitable for the use of transgenic fermentation in which canrenones were bioconverted to 11α-canned hydroxide.

Transformation was carried out in a Brown E biostart fermenter arranged as follows.

Volume; 15 L with round bottom

Height; 53 cm

diameter; 20 cm

H / D; 2.65

Impeller; 7.46 cm diameter, 6 pedals each 2.2 x 1.4 cm

Impeller space; 65.5, 14.5 and 25.5 cm from the bottom of the tank

baffler; 4 x 1.9 x 48 cm

Sparger; 21 holes with a diameter of 10.1 cm and ~ 1 mm

Temperature control; Provided by outer container cover

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

(Growth medium for bio-converting culture in 10 L fermentation tank) amount Quantity / L Glucose (amount after sterilization) 160 g 20 g peptone 160 g 20 g Yeast extract 160 g 20 g Antifoam SAF471 4.0 ml 0.5 ml It is made into 7.5L by an appropriate quantity of canlenone deionized water, and it sterilizes at 121 degreeC for 30 minutes. 160 g 20 g

The resulting suspension was stirred for 15 minutes, after which the volume was raised to 7.5 L with additional deionized water. The pH of the suspension was then adjusted to 5.2-6.5 by addition of a 20 wt% NaOH solution, after which the suspension was sterilized by heating at 121 ° C. for 30 minutes in a Brown E fermenter. The pH after sterilization was 6.3 ± 0.2 and the final volume was 7.0L. A sterile suspension was inoculated with some of the secondary species cultures of this example prepared as described above, and the volume was raised to 8.0 L by addition of 50% sterile glucose solution. Fermentation was carried out at 28 ° C. until PMV reached 50%, then lowered to 26 ° C. and lowered to 24 ° C. when PMV exceeded 50% to maintain a constant PMV of less than about 60%. . Air was injected through the sparger at a rate of 0.5vvm based on the initial liquid volume and the pressure in the fermenter was maintained at 700 milliga gauge. Agitation was started at 600 rpm and stepwise increased to 1000 rpm as needed to maintain a dissolved oxygen content of at least 30 vol%. Glucose concentrations were tracked. After the initial high glucose concentration fell below 1% by consumption by the fermentation reaction, supplemental glucose was provided with 50% by weight sterile glucose solution to maintain the concentration range in the range 0.05 to 1% throughout the remaining batch cycles. . The pH before inoculation was 6.3 ± 0.2. After the pH had dropped to about 5.3 during the initial fermentation period, it was maintained in the range of 5.5 ± 0.2 for the remaining cycles by the addition of ammonium hydroxide. Foaming was controlled by the addition of polyethylene glycol antifoam sold under the trade name SAG 471 by OSI Specialties, Inc.

The growth of the cultures occurred mainly during the first 24 hours of the cycle, with PMV of about 40%, pH of about 5.6 and dissolved oxygen content of about 50% by volume. Canrenone conversion started as the culture grew. The concentrations of carrenone and 11α-kanenone hydroxide were tracked during the bioconversion by analyzing the samples daily. The sample was extracted with hot ethyl acetate and the sample solution obtained was analyzed by TLC and HPLC. When the remaining canrenone concentration reached about 10% of the initial concentration, bioconversion was considered complete. Approximate conversion time was 110 to 130 hours.

When bioconversion was complete, the biomass of the mycelium was separated from the broth by centrifugation. The supernatant was extracted with the same volume of ethyl acetate and the aqueous layer was discarded. Mycelia fragments were resuspended in ethyl acetate using approximately 16 volumes per gram of canrenone filled with fermentation reactor. The mycelium suspension was redissolved for 1 hour under stirring, cooled to about 20 ° C. and filtered in a Buchner funnel. The filter cake of the mycelium was washed twice with 5 volumes of ethyl acetate per gram of canrenone in the fermenter, and washed with deionized water (1 L) to replace the remaining ethyl acetate. Aqueous extract, abundant solvent, solvent wash and water wash were mixed. The remaining extracted mycelium cake was discarded or reextracted according to the analysis for the remaining steroids therein. The mixed liquid phase was fixed for 2 hours. The aqueous phase was then separated off and the organic phase was concentrated until the remaining volume was approximately 500 ml. The bottom was then cooled to about 15 ° C. with slow stirring for 1 hour. The crystalline product was filtered off again by filtration and washed with ethyl acetate (100 ml). The solvent was removed from the crystal by evaporation and the crystalline material was dried under vacuum at 50 ° C.

Example 5

Lyophilized spores of Aspergillus ochraceus ATCC 18500 were suspended in corn steep liquor growth medium (2 ml) as described in Example 4. Ten agar plates were also prepared in the manner of Example 4. Plates were incubated and harvested as described in Example 4 to obtain master cell vanes. The vial containing the master cell vank was stored in the vapor phase of a liquid nitrogen cooler at -130 ° C.

From the vial of the master cell vane, an operating cell vane was prepared as described in Example 4 and stored in a nitrogen cooler at -130 ° C.

Growth medium (300 ml) having the composition shown in Table 19 was placed in a 2 L baffle flask. The working cell suspension was injected into the 3 mL flask. The inoculated mixture was incubated on a rotary shaker (200 rpm, 5 cm substitution) at 28 ° C. for 20-24 hours to produce main species cultures having a percent aggregated mycelium volume of approximately 45%. Visual inspection found that the cultures contained mycelium with a diameter of 1-2 mm, similar to pellets; Microscopic observation showed a pure culture.

Growth medium for primary and secondary inoculation cultures Quantity / L Glucose (amount after sterilization) 20 g peptone 20 g Yeast extract 20 g Make 1000 ml of an appropriate amount of distilled water. Sterilize at 121 ° C for 30 minutes.

Cultivation of secondary species cultures was started by injecting 8 L growth medium having the composition shown in Table 19 into a 14 L glass fermenter. 160 ml to 200 ml of the primary species culture of the present Example was inoculated into the fermenter. The preparation thereof is as described above. The inoculated mixture was incubated at 28 ° C. for 18-20 hours, stirred at 200 rpm, and the aeration rate was 0.5vvm. At the end of the proliferation, the culture showed the same properties as described above for the primary inoculation.

Transformation was carried out in a 60L fermenter substantially in the manner described in Example 4 except that the growth medium had the composition shown in Table 20 and the initial amount of secondary species culture was 350 ml to 700 ml. The stirring speed was initially 200 rpm, and was increased to 500 rpm as needed to maintain dissolved oxygen at 10 vol% or more. Approximate bioconversion time for 20 g / L canrenone was 80 to 160 hours.

Growth medium for bioconverting culture in 60 L fermenter amount Quantity / L Glucose (amount after sterilization) 17.5 g 0.5 g peptone 17.5 g 0.5 g Yeast extract 17.5 g 0.5 g Canrenone (amount as a 20% slurry in sterile water) 700 g 20 g Make an appropriate amount of deionized water at 35 L. Sterilize at 121 ° C for 30 minutes.

Example 6

Using spore suspensions from working cell vanes produced according to the manner described in Example 4, primary and secondary species cultures were also prepared substantially in the manner described in Example 4. Using the secondary species cultures produced in this manner, two bioconversion operations were performed according to a modified method of the type shown in FIG. 1 and two operations according to the method shown in FIG. 2. The transfection medium, canrenon addition method, harvest time and degree of conversion for this task are shown in Table 21. Operation R2A used the canrenone addition method based on the same principle as in Example 3, while operation R2C modified the method of Example 3 by only performing two canrenone additions, once at the beginning of the batch and once after 24 hours did. In operations R2B and R2C, the entire amount of canrenone was injected at the beginning of the batch, and as described in Example 4 except that the sterilized in a separate container and glucose was added as the batch progressed before the canrenone was placed in the fermenter. The reaction was carried out. A warping mixer was used to break up the large mass produced during sterilization. In operations R2A and R2B, canrenone was injected in a batch in methanol solution, in which the operation is more different than that of Examples 3 and 4.

Explanation of the Early Canrenon Biotransformation Process Job number R2A R2B R2C R2D Medium (g / l) Corn Steep Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH 3015315 Adjusted to 6.0 with 0.5 ml 2.5 N NaOH Same as R2A 30153300.5ml2.5N adjusted to 6.5 with NaOH Same as R2C Canrenon 10 g / 80 ml MeOH added at 0, 18, 24, 30, 36, 42, 50, 56, 62 and 68 hours Add 80 g / 640 ml MeOH all at once in 0 hours Sterilization and mixing 25 g at 0 hours; 200g added in 24 hours Add 200 g at 0 hours sterilization and mixing Collection time 143 hours. 166 hours. 125 hours. 104 hours. Conversion 45.9% 95.6% 98.1% 95.1%

In working R2A and R2B, the methanol concentration in the fermented beer was concentrated to about 6.0%, which was found to inhibit culture and bioconversion. However, based on the results of this work, fine particle sediments that help ethanol or other water-miscible solvents effectively increase the amount of canrenon at low concentrations and provide a large surface area for the supply of canlenone to ensure that the reaction occurs well As concluded that it helps to provide. Canrenones have been found to be stable at sterilization temperatures (121 ° C.) but are associated in lumps. A warping mixer was used to break up the mass into fine particles, which were successfully converted to the product.

Example 7

Using spore suspensions from working cell vanes produced according to the method described in Example 4, primary and secondary species cultures were also prepared substantially in the manner described in Example 4. The description and results of Example 7 are shown in Table 22. Using the secondary species cultures produced in this manner, one bioconversion (R3C) was performed substantially as described in Example 3, and three bioconversions were performed according to the method generally described in Example 5. Performed. In the latter three operations (R3A, R3B and R3D), canrenones were sterilized in a portable tank with growth medium excluding glucose. Glucose was aseptically supplied from another tank. Sterile canrenone suspensions were injected into the fermenter prior to inoculation or during the early stages of bioconversion. In task R3B, supplemented sterile canrenone and growth medium were injected at 46.5 hours. Agglomerates of canrenone produced during sterilization were pulverized through a waring mixer, thus producing a fine particle suspension to enter the fermenter. Transformation growth medium, canrenone addition method, nutrient addition method, harvest time and conversion for this task are shown in Tables 22 and 23.

Description of the Canrenon Biotransformation Process Job number R3A R3B R3C R3D Medium (g / l) Corn Steep Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH 3015315 Adjusted to 6.5 with 0.5 ml 2.5 N NaOH Same as R3A Peptone: 20 Yeast Extract: 20 Glucose: 20 OSA: 3ml Adjust to 6.5 with 2.5N NaOH Same as R3A Canrenone addition Canrenon was sterilized and mixed. BI: 50 g 16.5 h: 110 g Same as R3A BI: 50 g16.5 hours: 110 g46.5 hours: 80 g Non-Sterile Canrenones: Added by the Methods Listed in Table 23 Same as R3A BI: 50 g16.5 Hours: 110 g supply See Table 23 See Table 23 See Table 23 See Table 23 Collection time 118.5 hours. 118.5 hours. 118.5 hours. 73.5 hours. Conversion 93.7% 94.7% 60.0% 68.0%

Feeding Method for Canrenone, Glucose and Growth Medium in Growth Experiments Addition time (hours) R3C R3A R3B R3D Canrenon 200g / 2L sterilization DIg 50% solution of glucose 20g each in 20g 1L water of peptone and yeast extract 20 mg kanamycin 20 mg tetracycline 100 mg cephalexin in 50 ml of antibiotics Canrenon / Growth Medium (see Table 22) Canrenon / Growth Medium (see Table 22) Canrenon / Growth Medium (see Table 22) 0 50g / 0.4L 50g / 0.4L 50g / 0.4L 14.5 16 100 25 50 ml 16.5 110g / 1.2L 110g / 1.2L 110g / 1.2L 20.5 16 140 25 28.5 16 140 25 34.5 16 150 25 40.5 16 150 25 50 ml 46.5 880 130 25 80g / 0.8L 52.5 160 120 25 58.5 160 150 25 64.5 160 180 25 50 ml 70.5 160 140 25

Due to the growth of the islands, high viscosity fermented broth juice was seen in all four operations of this example. In order to overcome the obstacles that high viscosity creates with regard to aeration, mixing, pH control and temperature control, the aeration rate and stirring speed were increased during operation. The bioconversion proceeded satisfactorily under severe conditions, but a thick cake formed on the liquid juicy surface. Some unreacted canrenon was drowned out by this cake.

Example 8

The description and results of Example 8 are summarized in Table 24. Four fermenter operations in which 11α-canylene hydroxide was made by the bioconversion of canrenone were performed. In group 2 of these operations (R4A and R4D), bioconversion was carried out in substantially the same manner as the R3A and R3D operations of Example 6. In R4C, canrenone was converted to 11α-canannon hydroxide in the manner described in Example 3. The R4B operation was carried out by the method described in Example 4, ie sterilization of growth medium and canrenone in the fermenter immediately prior to inoculation, with a supplement solution containing all nitrogen and phosphate at the beginning of the batch and containing only glucose. Was injected into the fermenter to maintain glucose levels as the batch progressed. In the latter method (working R4B), glucose concentrations were tracked every 6 hours and added as directed to control glucose levels in the 0.5-1% range. The canrenon addition method for this operation is shown in Table 25.

Method Description Proliferative Experiment of Canrenone Bioconversion Job number R4A R4B R4C R4D Medium (g / l) Corn Steep Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH 3015315 Adjusted to 6.5 with 0.5 ml 2.5 N NaOH Same as R4A Peptone: 20 Yeast Extract: 20 Glucose: 20 OSA: 3ml Adjust to 6.5 with 2.5N NaOH Same as R4A Canrenone addition Canrenon was sterilized and mixed. BI: 40 g 23.5 h: 120 g 160 g canrenone was sterilized in a fermenter. Non-Sterile Canrenone: Table 25 Added by the Methods Listed Canrenone was sterilized and mixed. BI: 40 g 23.5 h: 120 g Discharge See Table 25 See Table 25 See Table 25 See Table 25 Collection time 122 hours. 122 hours. 122 hours. 122 hours. Conversion 95.6% 97.6% 95.4% 96.7%

How to Feed Canrenone, Glucose, and Growth Medium in Formula Experiments Addition time (hours) R4C R4A R4B R4D Canrenone 200g / 2L sterile water g 50% glucose solution g 20g each of 1L water of peptone and yeast extract 20 mg kanamycin 20 mg tetracycline 100 mg cephalexin in 50 ml of antibiotic (added to canrenone slurry) Growth medium (see Table 24) Growth medium (see Table 24) Growth medium (see Table 24) 14 600 135 25 50 ml 20 100 23 120g / 1.2L 120g / 1.2L 26 100 25 32 135 25 38 500 120 25 50 ml 44 100 25 50 100 25 56 150 25 62 500 150 25 50 ml 68 200 25 74 300 25 8- 100 25 86 125 25 92 175 25 98 150 104 175 110 175 116 200

All fermenters were run under high agitation and aeration during most of the fermentation cycles, as fermented beer became highly viscous immediately after inoculation or within one day.

Example 9

Transformation growth medium, canrenone addition method, harvest time and conversion degree for the leaves of this example are shown in Table 26.

Except as described below, four bioconversion operations were performed substantially in the manner described in task R4B of Example 8. In work R5B, the top turbine disk impeller, which was used to stir the other work, was replaced with a downward pumping marine impeller.

The pumping down to the shaft pushes the juice to the center of the fermenter and prevents cake formation. Methanol (200 ml) was added to task R5D immediately after inoculation. Since canrenone was sterilized in the fermenter, all nucleophiles except glucose were added at the beginning of the batch, eliminating the need for a chain supply of nitrogen sources, phosphorus or antibiotic sources.

Method Description Proliferation experiment of 10L bioconversion Job number R5A R5B R5C R5D Medium (g / l) Corn Steep Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH 3015315 Adjusted to 6.5 with 0.5 ml 2.5 N NaOH Same as R5A Peptone: 20 Yeast Extract: 20 Glucose: 20 OSA: 3ml Adjust to 6.5 with 2.5N NaOH Same as R5A Canrenone addition 160 g canrenone sterilized in fermenter 160g canrenone sterilized in fermenter 160 g canrenone sterilized in fermenter 160 g canrenone sterilized in fermenter Badge supply Glucose supply Glucose supply Glucose supply Glucose supply Collection time 119.5 hours 119.5 hours 106 119.5 hours Conversion 96% 94.1% 88.5% 92.4%

In order to maintain the solid immersion growing on the liquid surface, growth medium (2 L) was added to each fermenter 96 hours after the start of the batch. Although the mixing problem was not entirely overcome by the addition of growth medium or the use of a downward pumping impeller (Working R5B), the results of the work showed the possibilities and advantages of the method and a satisfactory mixing according to conventional practice. It has been shown that this can be provided.

Example 10

Three bioconversion operations were carried out substantially in the manner described in Example 9. Transformation growth medium, canrenone addition method, harvest time and conversion for the work of this example are shown in Table 27.

Explanation of proliferation experiment method of 10L bioconversion Job number R6A R6B R6C Medium (g / l) Corn Steep Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH 3015315 Adjusted to 6.5 with 0.5 ml 2.5 N NaOH Same as R6A Peptone: 20 Yeast Extract: 20 Glucose: 20 OSA: 0.5ml Adjust to 6.5 with 2.5N NaOH Canrenon sheep 160 g canrenone sterilized in fermenter 160g canrenone sterilized in fermenter 160 g canrenone sterilized in fermenter Badge supply Glucose feed; 1.3 L medium and 0.8 L sterile water at 71 hours Glucose supply; 0.5 L medium and 0.5 L sterile water at 95 hours Glucose supply; No other additions Collection time 120 hours 120 hours 120 hours Conversion 95% 96% 90% Mass Balance 59% 54% 80%

Growth medium (1.3 L) and sterile water (0.8 L) were added after 71 hours in operation R6A to immerse the mycelia cake grown on the surface of the liquid broth. For the same purpose, growth medium (0.5 L) and sterile water (0.5 L) were added after 95 hours in operation R6B. Material balance data showed that better mass balance can be determined when cakes stacked on the liquid surface are minimized.

Example 11

Fermentation was performed to compare the pre-sterilization of canrenone with the growth medium and sterilization of canrenone in the transgenic fermentor. In operation R7A, the reaction process was performed as shown in FIG. 2 under conditions comparable to the conditions of operations R2C, R2D, R3A, R3B, R3D, R4A, and R4D. Operation R7B was performed as shown in FIG. 3 under conditions comparable to those of Examples 4, 9 and 10, and operation R4B. The transgenic growth medium, canrenone addition method, harvest time and conversion for the work of this example are shown in Table 28.

10L scale biotransformation method description Job number R7A R7B Medium (g / l) Corn Steep Liquid Yeast Extract NH ₄ H ₂ PO ₄ Glucose OSApH 3015315 Adjusted to 6.5 with 0.5 ml 2.5 N NaOH Same as R7A Canrenone addition 160 g canrenone was sterile mixed outside the fermentation tank. 160 g canrenone was sterilized in a fermenter. Discharge Glucose supply; canrenone was added to a 1.6 L growth medium. Glucose supply; no other additions. Collection time 118.5 hours 118.5 hours Conversion 93% 89%

The mass balance based on the final sample taken from task R7B was 89.5%, indicating no significant substrate loss or degradation during bioconversion. Mixing was determined to be appropriate for both operations.

The residual glucose concentration was above 5-10 g desired per liter control range during the initial 80 hours. Work performance was clearly not affected by some cake accumulated in the upper space of both fermenters.

Example 12

Extraction efficiencies were determined in a series of 1L extraction operations summarized in Table 29. In each operation, steroids were extracted from the mycelium using ethyl acetate (1 L / L fermentation volume). Two extractions were performed in succession for each operation. Based on RP-HPLC, about 80% of the total steroid was recovered at the first extraction; The recovery was increased to 95% by the second extraction. In the third extraction, the remaining 3% of the steroids were recovered. The remaining 2% was lost from the supernatant. The extract was dried using vacuum but not washed with any additional solvent. If justified by the process economy, emissions to solvents will improve recovery from initial extraction.

 Recovery of 11α-canned hydroxide from 1 L extract (% of total) Job number 1st extraction Secondary extraction 3rd extraction Supernatant R5A 79% 16% 2% 2% R5A 84% 12% 2% 2% R4A 72% 20% 4% 4% R4A 79% 14% 2% 5% R4B 76% 19% 4% One% R4B 79% 16% 3% 2% R4B 82% 15% 2% One% Average 79% 16% 3% 2%

Methyl isobutyl ketone (MIBK) and toluene were assessed as extraction / crystallization solvents for 11α-kanenone hydroxide on a 1 L juicy scale. Using the extraction protocol described herein, MIBK and toluene could be compared with ethyl acetate in both extraction efficiency and crystallization performance.

Example 13

As part of the evaluation of the processes of FIGS. 2 and 3, particle size studies were performed on the canrenon substrate provided at the beginning of the fermentation cycle in each process. As mentioned above, the canrenone supplied by the process of FIG. 1 was pulverized before injecting into a fermenter. In this process, canrenone was not sterilized and the growth of unwanted microorganisms was controlled by the addition of antibiotics. In the processes of FIGS. 2 and 3, canrenone was sterilized before the reaction. In the process of FIG. 2 this is achieved in a mixer before injecting canrenone into the fermenter. In the process of FIG. 3, the suspension of canrenone in the growth medium was sterilized in the fermenter at the beginning of the batch. As discussed herein, sterilization tends to cause aggregation of canrenone particles. Because of the limited solubility of canrenones in aqueous growth media, the productivity of the process depends on the mass transfer from the solid phase, and thus can be expected to depend on the interfacial area presented by the solid particle substrate, which in turn depends on the particle size distribution. This consideration initially hindered the process of FIGS. 2 and 3.

However, the agitation in the blender of FIG. 2 and the fermentation tank of FIG. 3 is suitable for the size of the non-sterile and pulverized canrenone supplied to the process of FIG. 1 with the shear pump used for the movement of the batch of FIG. 2. It has been found to degrade in the particle size range. This is illustrated by the particle size distribution of canrenone at the beginning of each of the three in-process reaction cycles (see Table 30 and Figures 4 and 5).

Particle Distribution of Three Different Canrenon Samples Sample 45-125μ <180 μ Average diameter μ Job Number #:% Convert Canrenon 75% 95% - R3C: 99.1% (120 hours) R4C: 94.3% (120 hours) Mixed sample 31.2% 77.2% 139.5 R3A: 94.6% (120 hours) R3B: 95.2% (120 hours) Sterilized Sample 24.7% 65.1% 157.4 R4B: 97.6% (120 hours) R5B: 93.8% (120 hours)

From the data in Table 30, it was found that the agitation and shear pumps were effective to reduce the average particle diameter of sterilized canrenones in the same order of magnitude as the non-sterile substrate, but significant differences in size remained on the side of the non-sterile substrate. There will be. Despite this difference, the reactivity performance data showed that the presterilization performance was at least as productive as the process of FIG. Even more advantageous was recognized in the process of FIG. 2 by specific steps to further reduce and control the particle size, for example by pasteurization rather than wet grinding and / or sterilization of sterile canrenone.

Example 14

Species cultures were prepared in the manner described in Example 5. At 20 hours, the mycelium in the inoculum fermentation tank was fleshy at 40% PMV. Its pH ranges from 5.4 to 14.8 gpl and glucose remains unused.

Transformed growth medium (35 L) having the composition shown in Table 20 was prepared. During preparation of the feed medium, the glucose and yeast extracts were sterilized separately and mixed as a single feed at an initial concentration of 30 wt% glucose and 10 wt% yeast extract. The pH of this feed was adjusted to 5.7.

Using this medium (Table 20), two bioconversion operations were carried out for the conversion of canlenone to 11α-canylene hydroxide. Each operation was carried out in a 60 L fermenter equipped with a stirrer comprising one Luciton turbine impeller and two Lightnin 'A315 impellers.

The initial volume of growth medium for the fermenter was 35 liters. Unmilled and non-sterile canrenones were added at an initial concentration of 0.5%. The medium in the fermenter was inoculated with the seed cultures prepared in the manner described in Example 5 at an initial inoculation rate of 2.5%. The fermentation was carried out at a temperature of 28 ° C., agitation speed of 200-500 rpm, aeration rate of 0.5vvm and sufficient back pressure to maintain dissolved oxygen level of at least 20% by volume. Transgenic cultures grown during the production run were in the form of very small oval pellets (about 1-2 mm). Canrenone and supplemental nutrients were fed to the fermenter in the manner described in Example 1. Nutrient addition was done every 4 hours at a ratio of 3.4 g glucus and 0.6 g yeast extract per liter of juice in the fermenter.

In addition to the addition of glucose during the batch, the aeration rate, agitation rate, dissolved oxygen, PMV and pH during each operation of this example are shown in Table 31. Table 32 shows canrenon conversion profiles. Job R11A ended after 46 hours, job R11B continued for 96 hours. In the latter work, 93% conversion was achieved at 81 hours; Another feed addition was made at 84 hours; The supply was stopped. Note that a significant change in viscosity occurred between the time the supply was stopped and the end of the operation.

Fermentation R11A time Air (1pm) rpm % DO Back pressure PMV (%) pH Glucose concentration (g / L) 0.1 20 200 93 0 2 6.17 5.8 7 20 20 85.1 0 5 6.03 5.5 12.4 20 300 50.2 0 5.43 21.8 20 400 25.5 0 6.98 0 29 20 500 17 0 38 5.22 30.2 20 500 18.8 10 35 5.01 31 20 500 79 10 4.81 One 35.7 20 500 100 10 45 5.57 0 46.2 20 500 23 6 45 5.8 One Whole glucose: 27.5 g / l Whole yeast extract: 8.75 g / l Fermentation R11B time Air (1pm) rpm % DO Back pressure PMV (%) pH Glucose concentration (g / l) 0.1 20 200 92.9 0 2 5.98 5.4 7 20 200 82.3 0 5 5.9 5 12.4 20 300 49.5 0 5.48 21.8 20 400 18 0 40 7.12 0 29 20 500 36.8 0 35 5.1 3 35.7 20 500 94.5 10 4.74 0 46.2 20 500 14.5 6 45 5.32 2 55 20 500 16.7 10 5.31 0.5 58.6 20 500 19.4 15 5.32 One 61.9 20 500 13 15 40 5.36 2 71.7 20 500 13 15 42 5.37 0 81.1 20 500 22.9 15 5.42 2.5 85.6 20 500 22 15 45 5.48 One 97.5 20 500 108 15 45 6.47 0 117.7 20 500 15 7.38 0 Whole glucose: 63 g / l Whole yeast extract: 14.5 g / l

Fermentation R11A: Canrenone Conversion Concentration (g / l) Conversion rate Calculated OH-canrenone Switching speed (g / l / h) Sample time OH-canrenon Canrenon system (%) (g / l) Calculation Measure R11A-0 0.10 0.00 5.41 5.41 R11A-7 7.00 0.18 4.89 5.07 3.58 0.18 0.03 0.03 R11A-22 21.80 2.00 2.12 4.14 48.75 2.44 0.15 0.12 R11A-29 29.00 3.67 4.14 7.81 47.03 4.48 0.28 0.23 R11A-36 35.70 6.68 1.44 8.12 82.27 7.74 0.49 0.45 R11A-46 46.20 7.09 0.41 7.51 94.48 8.59 0.08 0.04 Fermentation R11B: Canrenone Conversion Concentration (g / l) Conversion rate Calculated OH-canrenone Switching speed (g / l / h) Sample time OH-canrenon Canrenon system (%) (g / l) Calculation Measure R11B-0 0.1 0.00 5.60 5.60 R11B-7 7.0 0.20 4.98 5.18 3.78 0.19 0.03 0.03 R11B-22 21.8 2.51 2.46 4.97 50.49 2.52 0.16 0.16 R11B-29 29.0 4.48 16.99 21.47 20.87 4.69 0.30 0.27 R11B-36 35.7 8.18 10.35 18.53 44.16 9.70 0.75 0.55 R11B-55 55.0 17.03 13.20 30.23 56.33 19.50 0.32 0.36 R11B-59 58.6 20.80 11.73 32.53 63.95 21.97 0.69 1.05 R11B-62 61.9 22.19 8.62 30.81 72.02 24.50 0.77 0.42 R11B-72 71.7 26.62 3.61 30.23 88.06 29.46 0.51 0.45 R11B-81 81.1 27.13 2.05 29.18 92.97 30.32 0.09 0.05 R11B-86 85.6 26.87 2.0. 28.88 93.02 30.11 -0.04 -0.06 R11B-97 97.5 23.95 1.71 25.66 93.34 30.22 0.01 -0.25 R11B-118 117.7 24.10 1.68 25.79 93.47 30.26 0.00 0.01

Example 15

Various cultures were tested according to the methods generally described above for the effectiveness of bioconversion to canaren's 11α-canalurenone.

Aspergillus niger ATCC 11394, Rhizopus arrhizus ATCC 11145 and Rhizopus stolonifer ATCC 6227b were prepared in the manner described in Example 5. The growth medium (50 ml) having the composition shown in Table 18 was inoculated with the spore suspension from a working cell van and placed in an incubator. Species cultures were prepared in an incubator by fermentation at 26 ° C. for 20 hours. The incubator was stirred at a speed of 200 rpm.

2 ml of each species of microorganism were used to inoculate the transfection flask containing the growth medium shown in Table 18. Each culture was used for inoculation of two flasks, all six. Canrenone (200 mg) was dissolved in methanol (4 ml) at 36 ° C, and 0.5 ml of this solution was injected into each flask. Bioconversion was performed daily with the addition of 50% by weight glucose solution and under the conditions described in Example 5. After the first 72 hours, the following observations were made of the growth of mycelia in each transgenic fermentation flask:

ATCC 11394-Good Homogeneous Growth

ATCC 11145-good growth in the first 48 hours, but mycelium agglomerated; There was no growth in the last 24 hours.

ATCC 6227b-good growth; mycelium sheep formation aggregated into clumps.

Juicy samples were taken and analyzed for degree of bioconversion. After 3 days, fermentation with ATCC 11394 provided a conversion of 80-90% to 11α-kanenone hydroxide; ATCC 11145 had 50% conversion; And ATCC 6227b provided 80-90% conversion.

Example 16

Using the method described in Example 15, additional microorganisms were tested for effectiveness in the conversion of canrenone to 11α-canylene hydroxide. The microorganisms tested and the test results are shown in Table 33:

Example 17

Various microorganisms were tested for effectiveness in the conversion of canrenone to 9α-canylene hydroxide. Fermentation medium for the work of this example was prepared as shown in Table 34.

Soy flour Dextrose 20 g Soy flour 5 g NaCl 5 g Yeast extract 5 g KH ₂ PO ₄ 5 g With water It is 1L. pH 7.0 Peptone / yeast extract / glucose Glucose 40 g Bactopeptone 10 g Yeast extract 5 g With water It is 1L. Muller-Hinton Milk juice 300 g Casamino acid 17.5 g Starch 1.5 g With water It is 1L.

Fungi were grown in soy flour medium and ketone yeast extract glucose, and atinomycetes and eubacteria were grown in soy flour (plus 0.9 wt% sodium formate prescribed for biotransformation) and muller- Grown in Hinton gravy.

Starting cultures were inoculated in cold spore colonies (20 ml soy flour in 250 Elenmeyer flasks). The flask was covered with a milk filter and a bioshield. Starting cultures (24 or 48 hours later) were used to inoculate -10% to 15% cross-sectional-metabolic medium (also 20 ml in 250 ml Elenmeyer flasks), the latter before addition of the steroid substrate for the transformation reaction. Incubate for 24 to 48 hours.

Canrenone was dissolved / suspended in methanol (20 mg / ml), sterile filtered and added to the culture at a final concentration of 0.1 mg / ml. All transgenic fermentation flasks were stirred at 250 rpm at room temperature, 26 ° C. and 60% humidity.

Biotransformation results were harvested at 5 and 48 or 24 hours after addition of the substrate. The harvest started with the addition of methylene chloride or ethyl acetate (23 ml) to the fermentor flask. The flask was then stirred for 2 minutes and the contents of each flask poured into a 50 ml conical tube. To separate the phases, the tubes were centrifuged at 4000 rpm for 20 minutes at room temperature. The organic phase from each tube was transferred to a 20 ml borosilicate glass vial and evaporated in fast vac. The vial was sealed and stored at -20 ° C.

To obtain the material for structure determination, bioconversion was raised to 500 ml by increasing the number of stirred flask fermentations to 25. At the time of collection (24 or 48 hours after addition of the substrate) ethyl acetate was added to each flask individually, the flask was sealed and placed again in the stirrer for 20 minutes. The contents of the flask were poured into polypropylene bottles and centrifuged to separate the phases, or poured into separation funnels in which the phases were separated by gravity. The organic phase was dried and a crude extract of the steroid contained in the reaction mixture was produced.

The reaction product was first analyzed by thin layer chromatography on silica gel (250 μm) on a fluorescent plate (254 nm). Ethyl acetate (500 μL) was added to each vial containing the dried ethyl acetate extract from the reaction mixture. Further analysis was performed by high performance liquid chromatography and mass spectroscopy. TLC plates were developed in a 95: 5 v / v chloroform / methanol solvent mixture.

Further analysis was performed by high performance liquid chromatography and mass spectroscopy. Waters HPLC with Millium software, photodiode array detector and automatic sampler was used. Reversed phase HPLC used a Waters NOVAPAK C-18 (4 μm particle diameter) RadialPak 4 mm cartridge. A 25 minute linear solvent gradient was started with the column initiated in water: acetonitrile (75:25) and terminated in water: acetonitrile (25:75). It was then washed for 4 minutes with a 3 minute gradient and isocratic in 100% acetonitrile before regenerating the column to initial conditions.

For LC / MS, ammonium acetate was added to both the acetonitrile phase and the aqueous phase at a concentration of 2 nM. The chromatography was not significantly affected. The eluate from the column was separated by 22: 1 and the mainstream of material was introduced into the PDA detector.

The remaining 4.5% of the material was introduced into the electrospray ionization chamber of the Sciex API III mass spectrometer. Mass spectroscopy was completed in positive mode. An analog data line from a PDA detector on HPLC transferred short wavelength chromatograms to a mass spectrometer for co-analysis of UV and MS data.

Mass spectroscopic crushing forms have been found to be useful for classification from hydroxylated substrates. Two expected hydroxylated canrenones, 11α-hydroxide and 9α-hydroxyl, released water at different frequencies in a certain way that could be used for diagnosis. In addition, 9α-kanenone hydroxide forms ammonium adducts more easily than 11α-kanenone hydroxide. A summary of TLC, HPLC / UV and LC / MS data for canrenon fermentation is shown in Table 35 and showed that the data of the tested microorganisms were effective in the bioconversion of 9len-hydroxynlenone hydroxide of canrenone. Among these, the preferred microorganism is Corynespora cassiicola ATCC 16718.

Example 18

Various cultures were tested for effectiveness in the bioconversion of androstenedione to 11α-androandrostendione according to the method described above.

Aspergillus ochraceus NRRL 405 (ATCC 18500); Aspergillus niger ATCC 11394; Aspergillus nidulans ATCC 11267; Rhizopus oryzae ATCC 11145; Rizopus stolonifer ATCC 6227b; Each of the working cell vanes of Tricotecium roseum ATCC 12519 and ATCC 8685 was prepared in the manner described in Example 4. Growth medium (50 ml) having the composition shown in Table 18 was inoculated with a suspension of spores from a cell cell van and placed in an incubator. Species cultures were prepared in an incubator by fermentation at 26 ° C. for about 20 hours. The incubator was stirred at a speed of 200 rpm.

2 ml of each of the microbial species cultures were used to inoculate the transforming flask containing the growth medium (30 ml) shown in Table 15. Each culture was inoculated in two flasks and used for all 16. Androstenedione (300 mg) was dissolved in methanol (6 ml) at 36 ° C., and 0.5 ml of this solution was injected into each flask. Bioconversion was performed under the conditions described in Example 6 for 48 hours. After 48 hours, the juicy samples were collected and extracted with ethyl acetate in the same manner as in Example 17. Ethyl acetate was concentrated by evaporation and the sample was analyzed by thin layer chromatography to determine if a material with a chromatographic flow similar to 11α-hydroxy-androstendione standard (Sigma Chimical Co., St. Louis) was present. did. The results are shown in Table 36. Positive results were marked with "+".

Bioconversion of Androstenedione to 11 Alpha-Hydroxy-Androstenedione Culture ATTC # badge TLC Results Rizopus orizaae (Rhizopus oryzae) 11145 CSL + Rhizopus stolonifer 6227b CSL + Aspergillus nidulans 11267 CSL + Aspergillus niger 11394 CSL + Aspergillus ochraceus NRRL 405 CSL + Aspergillus ochraceus 18500 CSL + Tricotesium roseum 12519 CSL + Tricotesium roseum 8685 CSL +

The data in Table 36 indicate that each of the listed cultures was capable of producing compounds from androstenedione with the same R _f value as the 11α-androstandionone standard.

Aspergillus ochraceus NRRL 405 (ATCC 18500) was tested again in the same manner as described above, and the cultures were separated and purified by normal silica gel column chromatography using methanol as solvent. Fragments were analyzed by thin layer chromatography. TLC plates were Whatman K6F silica gel 60 cc, 10 x 20 size, 250μ thick. The solvent mixture was chloroform: methanol, 95: 5, v / v. Both the crystallized material and the 11α-androstandione standard were analyzed by LC-MS and NMR spectroscopy. Both compounds yielded similar profiles and molecular weights.

Example 19A

Various microorganisms were tested for effectiveness in the bioconversion of androstenedione to 11β-hydroxyandrostendione by the method described above in Examples 17 and 18.

Aspergillus fumigatus ATCC 26934, Aspergillus niger ATCC 16888 and ATCC 26693, Epipicum oryzae ATCC 7156, Curularia lunata ATCC 12017 Individual cultures of Cunninghamella blakesleeana ATCC 8688a, and Pithomyces atro-olivaceus IFO 6651 were grown in the manner described in Example 17. Growth and fermentation medium (30 ml) has the composition shown in Table 34.

11β-hydroxylation of androstenedione by the microorganisms listed above was analyzed using the same identification method described in Examples 17 and 18. The results are shown in Table 19 A-1.

11β-hydroxylation of Androstenedione by Various Microorganisms Creature TLC LC / MS Aspergillus fumigatus ATCC 26934 (Aspergillus fumigatus) + + Aspergillus niger ATCC 1688 and ATCC 26693 (Aspergillus niger) + + Epicorcom Orizae ATCC 7156 (Epicoccum oryzae) + + Kuburaria lunata ATCC 12017 (Curvularia lunata) + + Cunninghamella blakesleeana (ATCC 8688a) + + Phytomyces atro-olivaceus IFO 6651 + + In Table 19A-1, "+" indicates the approximate exact molecular weight depending on the positive result, ie the R _f and LC / MS phases expected in thin layer chromatography.

delete

This result indicates that the listed microorganisms can carry out 11β-hydroxylation of androstenedione.

Example 19B

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

Fermentation conditions and analytical methods were the same as those in Example 17. TLC plate and solvent system are as described in Example 18. The principle of chromatographic analysis is as follows:

11α-mexanthone and 11α-kanenone hydroxide have the same chromatographic fluidity. 11α-canned and 9α-canrenone hydroxides exhibit the same flowable morphology as 11α-androandrostendione and 11β-androandrostendione. Therefore, 11α-mexanthone must have the same fluidity as 9α-canoxide. Therefore, the compound extracted from the growth medium is developed for 9α-canylene hydroxide as a standard. The results are shown in Table 36.

This data suggests that the majority of the organisms listed in this table produce the same or similar products of 11β-mexureron from mexrenone.

Example 19C

Various microorganisms were tested for effectiveness in the conversion of mexrenone to 11α-mexhydroxyl, Δ ^1,2 mexrenone, 6β-mexhydroxyl, 12β-mexhydrenone and 9α-mexhydroxyl. Mexrenone is a U.S. Patent No. of Weier, which is incorporated herein by reference. 3,787,396 and RMWeier et al., J. Med. Chem., Vol. 18, pp. 817-821 (1975). Fermentation broth was prepared as described in Example 17 except that mexrenone was included. Fermentation conditions were substantially the same as that in Example 17; The analytical method was also as described in Examples 17 and 18. TLC plate and solvent system were as described in Examples 17 and 18.

The microorganisms tested and the results obtained are shown in Table 19C-1.

Production of 11α-Mexxenone from Mexrenone by Various Microorganisms Creature TLC HPLC m / z 415: 399 Beauveria Bassiana ATCC 7159 (Beauveria bassiana) + + 5: 1 Beauveria Bassiana ATCC 13144 (Beauberia bassiana) + + 10: 1 Mortierella Isabella ATCC 42613 (Mortierella isabella) + + 1: 1 Cunninghamella blakesleeana (ATCC 8688a) + + 1: 1 Cumminghamella echinulata + + 1: 2 Cumminghamella elegans (Cumminghamella elegans) + + 1: 1 Absidia coerula ATCC 6647 (Absidia coerula) + + 1: 1 Aspergillus niger ATCC 16888 + + 4: 1 Gronronella Butieri ATCC 22822 (Gongronella butieri) + + 3: 1 Phytomyces atro-olivaceus ATCC 6651 + + 3: 1 Streptomyces hygroscopius ATCC 27438 (Streptomyces hygroscopicus) + + 3: 1

In Table 19C-1, "+" indicates a positive result, i.e., R _f expected in thin film chromatography and expected retention time in HPLC. m / z 417: 399 represents the maximum length ratio of 417 molecules (mexrenone hydroxide) to 399 molecules (mexrenone). The standard has a maximum length ratio of 10: 1 to m / z 417 to m / z 399. The product obtained from Beauveria bassiana ATCC 13144 was separated from the incubation mixture and analyzed by NMR, confirming that the structural profile by it was 11α-mex hydroxide. By analogy, the product obtained from the other microorganisms listed in Table 19C-1 was also estimated to be 11α-mexanthone.

Production of △ ^1,2 Mexrenone from Mexrenone by Various Microorganisms Creature m / z 399 HPLC TLC Rhodococcus equi (Rhodococcus equi) + + + Bacterium cyclo-oxydans ATCC 12673 + + + Comomonas testosteroni (ATCC 11996) + + + Nocardia Auretia ATCC 12674 (Nocardia aurentia) + + + Rhodococcus equi (Rhodococcus equi) + + +

In Table 19C-2, "+" indicates a positive result, i.e., R _f expected in thin film chromatography and expected retention time in HPLC.

The material obtained from Bacterium cyclo-oxydans ATCC 12673 was separated from the incubation mixture and separated by NMR, confirming that the structural profile was Δ ^1,2 mexrenone. By analogy, the product from the other microorganisms listed in Table 19C-2 was also estimated to be Δ ^1,2 mexrenone.

Production of 6β- and 12β-Mexrenone Hydroxide

Mortierella isabella ATCC 42613 was grown in the same manner as in Example 17 in the presence of mexrenone. Fermentation products were isolated and purified by flash chromatography. The purified product was analyzed by the same LC / MS, proton NMR and ¹³ C NMR as in Examples 17 and 18. The data show that the product contains 6β- and 12β-mexanthone.

Production of 9α-Mex Rexon from Mexrenone by Various Microorganisms Creature m / z 417 HPLC TLC Streptomyces hygroscopicus ATCC 27438 (Streptomyces hygroscopicus) + + + Gongronella Butleri ATCC 22822 (Gongronella butleri) + + + Cunninghamella blakesleeana (ATCC 8688a) + + + Cunninghamella echinulata (Cunninghamella echinulata) + + + Cunninghamella elegans (Cunninghamella elegans) + + + Mortierella Isabella ATCC 42613 (Mortierella isabellina) + + + Absidia coerula ATCC 6647 (Absidia coerula) + + + Beauveria Bassiana ATCC 7159 (Beauveria bassiana) + + + Beauveria Bassiana ATCC 13144 (Beauveria bassiana) + + + Aspergillus niger ATCC 16888 + + +

The microorganisms listed in Table 19C-3 were grown under the same conditions as in Example 17 in the presence of mexrenone. Fermentations were analyzed by TLC and LC / MS as in Examples 17 and 18. "+" Indicates a positive result, that is, the R _f expected in thin film chromatography and the retention time expected in HPLC. The data suggest that the product contains 9α-mexanthone.

Example 19D

Various microorganisms were tested for the effectiveness of the conversion of ^canrenone Δ ^9,11 - ^canrenone . Fermentation medium and growth conditions were essentially the same as in Example 17 except that canrenone was included in the medium. The assay method was as described in Examples 17 and 18. The microorganisms and the results are shown in Table 19D-1 below.

Conversion of ^canrenone to △ ^9,11 - ^canrenone by various microorganisms Creature m / z 339 HPLC TLC Bacterium cyclo-oxydans (Bacterium cyclo-oxydans) + + + Comomonas testosteroni (ATCC 11996) + + + Cylindrocarpon radicicola ATCC 11011 + + + Paecilomyces carneus ATCC 46579 + + + Septomymic affinis ATCC 6737 (Septomyxa affinis) + + + Rhodocus spp. ATCC 19070 (Rhodococcus spp.) + + +

Fermentations were analyzed by TLC and LC / MS as in Examples 17 and 18. "+" Indicates a positive result, i.e., R _f expected in thin layer chromatography and retention time expected in HPLC.

The product obtained from Comomonas testosteroni ATCC 11996 was isolated from the growth medium and analyzed by UV spectroscopy. The spectral profile confirms the presence of Δ ^9,11 -canrenone. By analogy, the product from the other microorganisms listed in Table 19D-1 was also estimated to be ^Δ9,11 - ^canrenone .

Example 20A

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

61.2 L (57.8 kg) of DMF and then 23.5 kg of 11-hydroxycanrenone 1 were charged into a 50-gallon glass-line reactor with stirring. 7.1 kg of lithium chloride was added to this mixture. Stir for 20 minutes and fill the mixture with 16.9 kg acetonecyanohydrin and then 5.1 kg triethylamine. Heated to 85 ° C. and held at this temperature for 13-18 hours. After the reaction, 353 L of water and then 5.6 kg of sodium bicarbonate were added. Cooled to 0 ° C. and transferred to a 200-gallon glass-line reactor and slowly cooled to 130 kg of 6.7% sodium hypochlorite. The product was filtered and washed with 3 × 40 L of water to give 21.4 kg of enamine product. H1 NMR (DMSO-d6): 7.6 (2H, bd), 4.53 (1H, d, J = 5.9), 3.71 (1H, m), 3.0-1.3 (17H, m), 1.20 (5H, m), 0.86 (3H, s), 0.51 (1H, t, J = 10)

Example 20B

7α-cyano-11α, 17-dihydroxy-3-oxo-17α-pregan-4-ene-21-carboxylic acid, γ-lactone Manufacture

50.0 g of 11-hydroxykanrenone and 150.0 mL of dimethylacetamide are placed in a clean, dry three-necked flask equipped with a mechanical stirrer, ripper, thermocouple and heating mantle, and 16.0 mL of sulfuric acid solution (50.0 mL of 98.7% Baker grade), prepared by mixing with 50.0 mL of water), was added to this mixture. Then a sodium cyanide solution consisting of 15.6 g sodium cyanide and 27.0 mL water was added.

The resulting mixture was heated at 80 ° C. for 7 hours and the conversion was checked periodically by TLC or HPLC. After about 7 hours, HPLC of the mixture showed the presence of 7-cyano compound. The mixture was stirred overnight and cooled to room temperature (about 22 ° C.). 200 mL of water and then 200 mL of methylene chloride were added to the mixture and the mixture of the two phases obtained was stirred and separated. The aqueous layer was a gel. 100 mL of sodium bicarbonate solution was added to the aqueous layer to break up the gel. Then discarded the water column.

The separated methylene chloride layer was washed with 100 mL of water and the mixture of the two phases obtained was stirred. Next, the methylene chloride layer separated by phase separation was separated into 200 g of silica gel (Aldrich 200-400 mesh, 60 Was filtered through). The filtrate was concentrated to 45 ° C. under reduced pressure using a water aspirator to dryness to give about 53.9 g of manuscript. The original product was dissolved in 50 mL of methylene chloride and treated with 40 mL of 4N hydrochloric acid in a separatory funnel to separate the two phases. The methylene chloride layer was washed with 50 mL of water. The combined aqueous layers were extracted with 50 mL of methylene chloride and the combined methylene chloride layers were dried over sodium sulfate to give 11α-hydroxykanrenone and the product 7α-cyano-11α, 17-dihydroxy-3-oxo-17α-pregne. 45 g of a solid, a mixture of -4-ene-21-carboxylic acid, γ-lactone, were provided.

A sample of the product was HPLC (column: 25 cm x 4.6 mm, 5μ altima C ₁₈ LL); Solvent gradient: solvent A = water / trifluoroacetic acid = 99.9 / 0.1, solvent B = acetonitrile / trifluoroacetic acid = 99.9 / 0.1, flow rate = 1.00 mL / min, gradient = 65:30 (v / v) ( A: B--initial), 35:65 (v / v) (after A: B--20 minutes), 10:90 (v / v) (after A: B--25 minutes); Analysis was performed by a diode array detector showing λ _max of 238 nm.

The reaction mixture was analyzed by HPLC-NMR under the following conditions: solvent from 75% D ₂ O, 25% acetonitrile to 25% D ₂ O, 75% acetonitrile over 25 minutes at a flow rate of 1 mL / min. HPLC-column using gradient: Zorbax RX-C8 (25 cm x 4.6 mm, 5μ); ¹ H NMR (obtained using WET solvent suppression): 5.84 (s, 1H), 4.01 (m, 1H), 3.2 (m, 1H), 2.9-1.4 (m, due to solvent compression of acetonitrile Insignificant integer), 0.93-0.86 (s, overlapping 3H, and t, 2H).

Example 20C

5β, 7α-dicyano-17-hydroxy-3-oxo-17α-pregnan-21-carboxylic acid, γ-lactone Manufacture

102 g (0.3 moles) of 17-hydroxy-3-oxo-17α-pregna-4,6-diene-21-carboxylic acid and γ-lactone (canrenone) in 3 liters, 46.8 in a three-neck, round bottom flask g (0.72 moles) potassium cyanide, 78,6 mL (1.356 moles) acetic acid, and 600 mL methanol. 64.8 mL (0.78 mole) of pyrrolidine was added to the mixture and the combined slurry was heated to reflux (64 ° C.) and maintained for about 1.5 hours. The temperature of the slurry was lowered from 25 ° C. to 30 ° C. with a cooling bath over 10 minutes. 120 mL of concentrated hydrochloric acid was added slowly during cooling to precipitate a tan solid.

The mixture was stirred at 25 ° C. to 30 ° C. for 1.5 hours, after which additional 500 mL of water was added within 30 minutes. The mixture was cooled to 5 ° C. in an ice bath and 100 mL of aqueous 9.5 M sodium hydroxide (0.95 mol) was added to adjust the pH to 3 to 5.5 (monitored using a pH strip). Excess cyanide was removed by adding household bleach. 25 mL (0.020 mol) was added to achieve a negative starch iodide test. The cold mixture (10 ° C.) was filtered and the solid was washed with water until the rinse showed neutral pH (pH strip). The solid was dried at 60 ° C. to yield 111.4 g of solid.

The separated solid was melted at 244 ° C. to 246 ° C. on Fisher Johns blocks. The methanol solution containing a solid did not show any absorbance in the UV region of 210 to 240 nm. IR (CHCl ₃ ) cm ⁻¹ 2222 (cyanide), 1775 (lactone), 1732 (3-keto). ¹ H NMR (pyridine d ₅ ) ppm 0.94 (s, 3H), 1.23 (s, 3H).

Example 21A

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

50 kg of enamine 2 , about 445 L of 0.8N dilute hydrochloric acid and 75 L of methanol were charged into a 200-gallon glass-line reactor. The mixture was heated to 80 ° C. for 5 hours and cooled to 0 ° C. for 2 hours. The solid product was filtered to give 36.5 kg of dry product diketone. H ¹ NMR (DMSO-d ₆ ): 4.53 (1H, d, J = 6), 3.74 (2H, m), 2.73 (1H, doublet of doublets, J = 14, 7) 2.65-2.14 (8H, m), 2.05 (1H, t, J = 11), 1.98-1.71 (4H, m), 1.64 (1H, m), 1.55 (1H, dd, J = 13, 5), 1.45-1.20 (7H, m), 0.86 ( 3H, s).

Example 21B

Scheme 1: steps 1 and 2: 4 ′S (4′α), 7′α-hexadecahydro-11′α-hydroxy-10′β, 13′β-dimethyl-3 ′ from 11α-hydroxycanrenone , 5,20'-Trioxose pyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbo In situ preparation of nitrile

100 g (280.54 mmol) of 11-hydroxycanrenone, prepared in the same manner as in Example 1, followed by 300 mL of dimethylacetamide (Aldrich) into a reactor equipped with a cooling chuck, a mechanical stirrer, a heating mantle and a controller, and a funnel Filled up. The mixture was stirred until 11-hydroxycanrenone was dissolved. 31.5 mL of 50% sulfuric acid (Fisher) was added to this mixture to raise the temperature of the resulting mixture to 10 ° C to 15 ° C. Then, sodium cyanide solution prepared by dissolving 31.18 g (617.20 mmol) of sodium cyanide in 54 mL of deionized water was added to the 11α-hydroxycanrenone mixture over 2 to 3 minutes. The temperature of the obtained mixture rose to about 20 ° C to 25 ° C after the addition of sodium cyanide solution.

The mixture was heated to 80 ° C. and maintained at this temperature for 2-3 hours. HPLC analysis, once showing the conversion of 11α-hydroxykanrenone to enamine, was generally complete (above 98% conversion) and the heat source was removed. Without separating the enamine contained in the mixture, 148 mL of 50% sulfuric acid was added over an additional 3-5 minutes. Then 497 mL of deionized water was added to the mixture over 10 minutes.

The mixture was heated to 102 ° C. and maintained at that temperature until about 500 g of distillate was removed from the mixture. During the reaction / distillation, 500 mL of deionized water was added to the mixture in four separate 125 mL portions. Each portion was added to the mixture after equivalents of distillate (about 125 mL) were removed. The mixture was cooled to about 80 ° C. over 20 minutes when HPLC analysis indicated that the hydrolysis reaction from enamine to diketone was largely over (at least about 98%).

The mixture was filtered through a glass funnel. The reactor was rinsed with 1.2 L deionized water to remove the remaining product. The solid on the filter was washed three times using an equivalent amount of rinse water (approximately 0.4 L). A 1 L methanol and deionized water (1: 1 v / v) solution was prepared in the reactor and the filtrate was washed with 500 mL of this solution. The filtrate was then washed second with the remaining 500 mL of methanol / water solution. The vacuum was funneled to dry the filtrate sufficiently for transfer. The filtrate was transferred to a dry oven to be dried under vacuum for 16 hours and 84 g of dry product diketone, 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β -Dimethyl-3 ', 5,20'-trioxospyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2 'H) -carbonitrile was obtained. HPLC quantitation showed 94% of the desired diketone.

Example 22

Scheme 1: Step 3A: Method A: Preparation of methyl hydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

A four-necked 5L bottom flask was equipped with a mechanical stirrer, a pressure equalizing funnel with nitrogen inlet tube, a thermometer and a chuck with a bubbler. The bubbler was connected to two 2-L traps via tigon tubing, and the first one was emptied to prevent backflow of the material in the second trap (1 L sodium hypochlorite) into the reaction vessel. Diketone 3 (79.50 g; [Weight not corrected to purity, purity 85%]) was placed in a flask of 3 L methanol. A 25% methanolic sodium methoxide solution (64.83 g) was placed in the funnel and added dropwise with stirring under nitrogen over 10 minutes. After the dropping was completed, the reaction mixture of yellow near orange was heated to reflux for 20 hours. After this period, 167 mL of 4N HCl (note: HCN generated here!) Is added dropwise through an addition funnel to obtain a stopped reflux reaction mixture. The reaction mixture was lightened to a faded orange orange. The ridges were then replaced with take-offheads and 1.5 L of methanol was removed by distillation and 1.5 L of water was added to the flask simultaneously via funnel at the rate of distillation. The reaction mixture was cooled to ambient temperature and extracted twice with an aliquot of 2.25 L of methylene chloride. The combined extracts were washed successively with an aliquot of 750 mL cold saturated NaCl solution, 1N NaOH and again saturated NaCl. The organic layer is dried over sodium sulfate overnight, filtered and reduced to ˜250 mL in vacuo. Toluene (300 mL) was added and the remaining methylene chloride was stripped under reduced pressure during the time when the white solid product began to form on the wall of the flask. The contents of the flask were cooled overnight and the solids were removed by filtration. Washed with 250 mL of toluene, washed twice with 250 mL ether aliquots and dried on a vacuum funnel to give 58.49 g of a white solid which was 97.3% pure by HPLC. The mother liquor was concentrated to give an additional 6.76 g of purity 77.1% product. The total yield adjusted to purity was 78%.

H ¹ NMR (CDCl ₃ ): 5.70 (1H, s), 4.08 (1H, s), 3.67 (3H, s), 2.9-1.6 (19H, m), 1.5-1.2 (5H, m), 1.03 (3H , s).

Example 23

Scheme 1: step 3B: methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone methylhydrogen 17α-hydroxy-11α- (methyl Sulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, conversion to γ-lactone.

The 5-L four-necked flask is installed as in the example above except that no trapping system is installed except the bubbler. A quantitative amount of 138.70 g of hydroxyester was added followed by 1425 mL of methylene chloride to the flask with stirring under nitrogen. The reaction mixture was cooled to -5 ° C using salt / ice bath. Methanesulfonyl chloride (51.15 g, 0.447 mol) was added quickly, and triethylamine (54.37 g) in 225 mL methylene chloride was slowly added dropwise by addition. The dropping process, which required ˜30 minutes, was controlled so that the reaction temperature did not rise to about 5 ° C. After dropping, stirring was continued for 1 hour and the reaction contents were transferred to a 12-L separatory funnel, to which 2100 mL of methylene chloride was added. The solution was washed successively with 700 mL each aliquot of cold 1N HCl, 1N NaOH, and aqueous saturated NaCl solution. Aqueous water cleaners were combined and back extracted with 3500 mL methylene chloride. All organic cleaners were combined in a 9-L jug and 500 g of neutral alumina, active class II, and 500 g of anhydrous sodium sulfate were added thereto. The contents of the zerg were mixed well for 30 minutes and filtered. The filtrate was dried in vacuo to give a sticky yellow foam. It was dissolved in 350 mL of methylene chloride and added dropwise with stirring of 1800 mL of ether. The dropping rate was adjusted to add about 1/2 of ether over 30 minutes. After addition of about 750 mL, the product began to separate into a crystalline solid. The remaining ether was added within 10 minutes. The solid was removed by filtration and the filter cake was washed with 2 L of ether and dried overnight in a vacuum oven at 50 ° C. to 144.61 g (88%) of nearly white solid, m.p. 149 ° C-150 ° C was obtained. Materials prepared in this manner are typically 98-99% pure by HPLC (area%). A single run yielded a material having a melting point of 153 ° C.-153.5 ° C. and a purity of 99.5% determined by HPLC region.

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

Example 24

Scheme 1: Step 3C: Method A: Preparation of 7-methyl hydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

A 1-L four-necked flask was installed as in the second example. Formic acid (250 mL) and acetic anhydride (62 mL) were added to the flask with stirring under nitrogen. Potassium formate (6.17 g) was added and the reaction mixture was heated in an oil bath for 16 hours until the internal temperature reached 40 ° C. (which was subsequently repeated at 70 ° C. for better results). After 16 h, mesylate was added to increase the internal temperature to 100 ° C. Heating and stirring were continued for 2 hours, after which the solvent was removed in vacuo with rotabab. The remainder was stirred with 500 mL of ice water for 15 minutes and then extracted twice with 500 mL aliquots of ethyl acetate. The organic phases were combined and washed successively with cold 250 mL aliquots of saturated sodium chloride solution (twice), 1N sodium hydroxide solution, and again saturated sodium chloride. The organic phase was then dried over sodium sulphate, filtered and vacuum dried to yield a white foam that was close to yellow, which was pulverized into a glassy material when patted lightly with a spatula. 14.65 g of powder formed were 82.1% 7-methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone; 7.4% 7-Methylhydrogen 17α-hydroxy-3-oxopregna-4, 11-diene-7α, 21-dicarboxylate, γ-lactone; And 5.7% 9α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, bis (γ-lactone) (by HPLC region%). ).

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

Example 25

Scheme 1: Step 3C: Method B: Preparation of methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

A 5 L four-necked flask was set up as above example and 228.26 g acetic acid and 41.37 g sodium acetate were added with stirring under nitrogen. Using an oil bath, the mixture was heated to an internal temperature of 100 ° C. Mesylate (123.65 g) was added and heating continued for 30 minutes. At the end of this time, heating was stopped and 200 mL of ice water was added. The temperature dropped to 40 ° C. and stirring was continued for 1 hour, after which the reaction mixture was slowly poured into 1.5 L of cold water in a 5 L stirred flask. The product was separated into sticky oil. The oil was dissolved in cold 1 L ethyl acetate, respectively, and washed with 1 L saturated sodium chloride solution, 1 N sodium hydroxide, and finally saturated sodium chloride again. The organic phase was dried over sodium sulfate and filtered. The filtrate was dried in vacuo to give a foam that weakened with sticky oil. It was some time polished with ether and subsequently solidified. The solid was filtered and washed with excess ether to give 79.59 g of a yellowish white solid. It consists of 70.4% of the desired Δ ^9,11 ester 6 , 12.3% of Δ ^11,12 ester 8 , 10.8% of 7-α, 9-α-lactone 9 and 5.7% unreacted 5 .

Example 26

Scheme 1: Step 3D: Method A: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

A 500 mL reactor with four jackets was equipped with an addition funnel with a mechanical stirrer, ripper / bubble, thermometer and nitrogen inlet tube. The reactor was charged with 8.32 g of one-ene ester in 83 mL methylene chloride while stirring under nitrogen. To this was added 4.02 g dibasic potassium phosphate followed by 12 mL of trichloroacetonitrile. External cooling water was flowed through the reactor jacket to cool the reaction mixture to 8 ° C. 36 mL of 30% hydrogen hydroxide was added over 10 minutes in the addition funnel. The initial faded yellow reaction mixture turned almost colorless after addition. The reaction mixture remained at 9 ± 1 ° C. during the addition and continued stirring overnight (total 23 hours). Methylene chloride (150 mL) was added to the reaction mixture and all the contents were placed in ˜250 mL of ice water. It was extracted three times with 150 mL of methylene chloride aliquots. The combined methylene chloride extract was washed with 400 mL cold 3% sodium sulfite solution to decompose any remaining hydrogen peroxide. Then 330mL cold 1N sodium hydroxide wash, 400mL cold 1N hydrochloric acid wash, and finally wash with 400mL brine. The organic phase was dried over magnesium sulfate, filtered and the filter cake was washed with 80 mL of methylene chloride. The solvent was removed in vacuo to yield 9.10 g faded yellow solid product. It was recrystallized from ˜25 mL 2-butanone to give 5.52 g of nearly white crystals. The final recrystallization with acetone ~ 50 mL to obtain a long, needle-like crystals of mp 241-243 ℃, 3.16g.

H ¹ NMR (CDCl ₃ ): 5.92 (1 H, s), 3.67 (3 H, s), 3.13 (1 H, d, J = 5), 2.89 (1 H, m), 2.81-2.69 (15 H, m), 1.72 (1H, doublet of doublets, J = 5,15), 1.52-1.22 (5H, m), 1.04 (3H, s).

Example 27

Scheme 1: Step 3: Choice 1: 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tree Methylhydrogen 9,11α from oxospyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile With epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

The diketone (20 g) was charged to a clean dry reactor followed by the addition of 820 mL of MeOH and 17.6 mL of 25% NaOMe / MeOH solution. The reaction mixture was heated to reflux (˜67 ° C.) for 16-20 hours. The product was quenched with 40 mL of 4N HCl. The solvent was removed by distillation at atmospheric pressure. 100 mL of toluene was added and the remaining methanol was removed by azeotropic distillation with toluene. After concentration, raw hydroxyester 4 was dissolved in 206 mL of methylene chloride and cooled to 0 ° C. Methanesulfonyl chloride (5 mL) and then 10.8 mL of triethylamine were added slowly. The product was stirred for 45 minutes and the solvent was removed by vacuum distillation to give one mesylate 5 .

5.93 g of potassium formate, 240 mL of formic acid and then 118 mL of acetic anhydride were added to the separation drying reactor. The mixture was heated to 70 ° C. for 4 hours.

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

The product was dissolved in 180 mL of methylene chloride and cooled to 0-15 ° C. 8.68 g dipotassium hydrogen phosphate was added followed by 2.9 mL trichloroacetonitrile. A 78 mL solution of 30% hydrogen peroxide was added to the mixture over 3 minutes. The reaction mixture was stirred at 0-15 ° C. for 6-24 hours. After the reaction, the mixture of two phases was separated. The organic layer was washed with 126 mL of 3% sodium sulfite solution, 126 mL of 0.5N sodium hydroxide solution, 126 mL of 1N hydrochloric acid, and 126 mL of 10% brine. The product was dried over anhydrous magnesium sulfate or filtered through Celite and the solvent methylene chloride was removed by distillation at atmospheric pressure. The product was recrystallized twice with methyl ethyl ketone to give 7.2 g of epoxyxenone.

Example 28

Scheme 1: Step 3: Choice 2: 1'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-tree Methylhydrogen 9,11α of oxospyro [furan-2 (3H), 17'β- [4,7] methano [17H] cyclopenta [a] phenanthrene] -5'β (2'H) -carbonitrile No intermediate separation to epoxy-17α-hydroxy-3-oxopregin-4-ene-7α, 21-dicarboxylate, γ-lactone.

A 4-neck 5-L round bottom flask was equipped with a mechanical stirrer, an addition funnel with a nitrogen inlet tube, a thermometer, and a ripper with a bubbler attached to a sodium hypochlorite scrubber. Diketone (83.20 g) was placed in a flask with 3.05 L methanol. The addition funnel was charged with 67.85 g of 25% (w: w) sodium methoxide solution in methanol. The methoxide was added dropwise to the flask over 15 minutes with stirring under nitrogen. A dark orange / yellow slurry appeared. The reaction mixture was heated to reflux for 20 hours and 175 mL 4N hydrochloric acid was added dropwise during reflux (caution, HCN generated during this operation!). The reflux shaft was replaced with a take-off head and 1.6 L of methanol was removed by distillation while 1.6 L of 10% aqueous sodium chloride solution was added through the funnel at a rate consistent with the distillation rate. The mixture was cooled to ambient temperature and extracted twice with an aliquot of 2.25 L of methylene chloride. The combined extracts were washed with cold 750 mL 1N sodium hydroxide and saturated sodium chloride solution. The organic layer was dried by azeotropic distillation of methanol at 1 atm, resulting in a volume of 1 L (0.5% of the total was removed for analysis).

The concentrated organic solution (hydroxyester) was added back to the original reaction flask installed as before but there was no HCN trap. The flask was cooled to 0 ° C. and 30.7 g methanesulfonyl chloride was added while stirring under nitrogen. The addition funnel was added dropwise with 32.65 g trimethylamine over 15 minutes and the temperature was maintained at 5 ° C. Agitation was continued for 2 hours and the reaction mixture was warmed to ambient temperature. A column consisting of 250 g Dowex 50W × 8-100 acid ion exchange resin was prepared and washed with 250 mL water, 250 mL methanol and 500 mL methylene chloride before use. The reaction mixture was flowed into this column and recovered. A clean column was prepared and the above procedure was repeated. A third 250 g column consisting of Dowex 1 × 8-200 base ion exchange resins was prepared and pretreated as described above for the acid balance treatment. The reaction mixture was flowed into this column and recovered. Each column pass was subjected to two 250 mL methylene chloride washes below the column, each pass taking ˜10 minutes. Solvent washing was combined with the reaction mixture and the volume was reduced to ˜500 mL in vacuo and 2% of this was removed for qc. The remaining ones were further reduced to yield a volume of 150 mL (one mesylate solution).

960 mL formic acid, 472 mL acetic anhydride and 23.70 g potassium formate were placed in the original 5-L reaction setup. The mixture was heated with stirring at 70 ° C. under nitrogen for 16 h. The temperature was then increased to 100 ° C. and the raw mesylate solution was added over 30 minutes via an addition funnel. The temperature dropped to 85 ° C. while methylene chloride was distilled out of the reaction mixture. After all methylene chloride had been removed, the temperature rose again to 100 ° C. and maintained for 2.5 hours. The reaction mixture was cooled to 40 ° C. and formic acid was removed under pressure until the minimum stirring volume (˜150 mL) was reached. The rest was cooled to ambient temperature and 375 mL methylene chloride was added. The diluted residue was washed with a portion of cold 1N saturated sodium chloride solution, 1N sodium carbonate and again sodium chloride solution. The organic phase was dried over magnesium sulphate (150 g) and filtered to give a brown solution (one-ester ester solution) close to dark red.

A 1 L reactor with a four-neck jacket was installed with a mechanical stirrer, ripper / bubble, thermometer and addition funnel with nitrogen inlet tube. The reactor was filled with a solution of one-ester ester in 600 mL methylene chloride (measured at 60 g) with stirring under nitrogen. To this was added 24.0 g dibasic potassium phosphate followed by 87 mL trichloroacetonitrile. External cold water was flowed through the reactor jacket to cool the reaction mixture to 10 ° C. 147 mL 30% hydrogen peroxide was added to the mixture over 30 minutes in the addition funnel. The initial dark reddish brown reaction mixture turned a faded yellow after the addition was complete. The reaction mixture remained at 10 ± 1 ° C. during addition and continued stirring overnight (total 23 hours). The phases were separated and the aqueous portion was extracted twice with a portion of 120 mL methylene chloride. The combined organic phases were then washed with 210 mL 3% sodium sulfite solution and repeated twice, and both the organic and water soluble portions were negative for peroxide according to the starch / iodine test paper. The organic phase was washed successively with 210 mL aliquots of cold 1N sodium hydroxide, 1N hydrochloric acid and finally with brine twice. The organic phase was azeotropically dried to make a volume of ˜100 mL, 250 mL of clean solvent was added and azeotropic distillation to make the same 100 mL, and the remaining solvent was removed in vacuo to give 57.05 g of the product as a tacky yellow foam. A portion (51.01 g) was further dried to give a 44.3 g dose and quantified by HPLC. Quantified by 27.1% epoxymexrenone.

Example 29 Formation of 3-Epoxy-11α-hydroxy-Androstar-3,5-Dien-17-one from 11α-hydroxyandrostenedione

11α-hydroxyandrostenedione (429.5 g) and toluenesulfonic acid hydrate (7.1) were charged to the reaction flask under nitrogen. Ethanol (2.58 L) was added to the reactor and the resulting solution was cooled to 5 ° C. Triethylorthoformic acid (334.5 g) was added to the solution at 0 ° C. to 15 ° C. over 15 minutes. After addition of triethylorthopractic acid the reaction mixture was warmed to 40 ° C. and reacted at that temperature for 2 hours, then the temperature was raised to reflux and the reaction continued at reflux for an additional 3 hours. The reaction mixture was cooled in vacuo and the solvent removed in vacuo to afford 3-ethoxy-11α-hydroxy-androstar-3,5-diene-17-one.

Example 30 Formation of Enamine from 11α-hydroxykanrenone

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

Sodium cyanide (1.72 g) was placed in a 25 mL three neck flask equipped with a mechanical stirrer. Water (2.1 mL) was added and the mixture was stirred while heating until the solid dissolved. Dimethylformamide (15 mL) was added followed by 11α-hydroxykanrenone (5.0 g). A mixture of water (0.4 mL) and sulfuric acid (1.49 g) was added to the mixture. The mixture was heated to 85 ° C. for 2.5 h where HPLC analysis indicated complete conversion to the product. The reaction mixture was cooled to room temperature. Sulfuric acid (0.83 g) was added and the mixture was stirred for half an hour. The reaction mixture was poured into 60 mL of water and cooled in an ice bath. The flask was washed with 3 mL DMF and 5 mL water. The slurry was stirred for 40 minutes and filtered. The filter cake was washed twice with 40 mL water and dried at 60 ° C. overnight in a vacuum oven to give 11α-hydroxyenamine, etc. 5R '(5'α), 7'β-20'-aminohexadecahydro-11'β- Hydroxy-10'a, 13'α-dimethyl-3 ', 5-dioxospyro [furan-2 (3H), 17'α (5'H)-[7,4] metheno [4H] cyclopenta [a] phenanthrene] -5'-carbonitrile (4.9 g) was obtained.

Example 31-One-pot Conversion of 11α-hydroxykanrenone to Diketone

Sodium cyanide (1.03 g) was placed in a 50 mL three neck flask equipped with a mechanical stirrer. Water (1.26 mL) was added and the flask was slightly heated to dissolve the solids. Dimethylacetamide [or dimethylformamide] (9 mL) was added followed by 11α-hydroxycanrenone (3.0 g). A mixture of sulfuric acid (0.47 mL) and water (0.25 mL) was added to the reaction flask during stirring. Additional water (25 mL) and sulfuric acid (0.90 mL) were injected and the reaction mixture was stirred for 16 h. The mixture was then cooled to 5-10 ° C. in an ice bath. The solid was separated by filtration through a sintered glass strainer and then washed twice with water (20 mL). Solid diketone, such as 4'S (4'α), 7'α-hexadecahydro-11'α-hydroxy-10'β, 13'β-dimethyl-3 ', 5,20'-trioxospyro [furan- 3.0 g of solids were dried by vacuum drying 2 (3H), 17'β- [4,7] methano [17H] cyclo-penta [a] phenanthrene] -5'β (2'H) -carbonitrile in a vacuum oven. Got.

Example 32A-1

Scheme 1: Step 3A: Method B: Preparation of methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone. A suspension of 5.0 g of diketone in methanol (100 ml) produced by the method was heated to dissolve again and a 25% solution of calcium methoxide in methanol (5.8 ml) was added over 1 minute. Precipitation occurred after 15 minutes. The mixture was heated to dissolve and became homogeneous again after about 4 hours. Heating to dissolve was continued for 23.5 hours and 4.0N HCl (10 ml) was added. All 60 ml of hydrogen cyanide solution in methanol were removed by distillation. Water (57 ml) was added to the distillation residue over 15 minutes. The temperature of the solution was raised to 81.5 ° C. when water was added and 4 ml of additional hydrogen cyanide / methanol solution was removed by distillation. At the end of the water addition, the mixture was sprinkled and the heat supply was removed. The mixture was stirred for 3.5 hours to produce crystals slowly. The suspension was filtered, the collected solid was washed with water, dried with a stream of air on a funnel and dried at 92 ° C. for 16 hours to give 2.98 g of a gray solid. The solid was 91.4 weight% of hydroxyester, namely methyl hydrogen 11α, 17α-dihydroxy-3-oxoprene-4-ene-7α, 21-dicarboxylate, γ-lactone. The yield was 56.1%. Example 31A-2 Preparation of Methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

The diketone (40 g) prepared by the method described in Example 31 is placed in a clean, dry, jacketed 1 L reactor equipped with a bottom drain, ripper, RTD probe and fraction recovery receiver. Methanol (800 mL) is then placed in the reactor and the mixture is stirred. The resulting slurry is heated to about 60 ° C. to 65 ° C. and 25% potassium methoxide solution (27.8 mL) is added. The mixture is homogenized.

The mixture is heated at reflux. After about 1.5 hours of heating at reflux, 16.7 mL of 25% potassium methoxide solution is added to the mixture while maintaining at reflux. The mixture is kept at reflux for an additional 6 hours. Conversion of diketone to hydroxyester is analyzed by HPLC. Once HPLC shows that the ratio of diketone to hydroxyester is less than about 10%, 77 mL of 4M HCl (which can replace hydrochloric acid with a significant amount of 1.5M to 3M sulfuric acid) for about 15 minutes To the mixture over time.

The mixture is distilled off and about 520 mL of methanol / HCN distillate is recovered and discarded. The concentrated mixture is cooled to about 65 ° C. About 520 mL of water is placed in the mixture for about 90 minutes and maintained at about 65 ° C. while adding temperature. The mixture is gradually cooled to about 15 ° C. over about 4 hours, then stirred and held at about 15 ° C. for an additional 2 hours. The mixture is filtered and the filtered products are washed twice with about 200 mL of water each. The filtered product is dried in vacuo (90 ° C., 25 mm Hg). About 25 to 27 g of a off-white solid consisting mainly of methylhydrogen 11α, 17α-dihydroxy-3-oxopreg-4-ene-7α, 21-dicarboxylate, γ-lactone are obtained.

Example 32B-1

7-methyl hydrogen 5β-cyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone Manufacture

The reaction flask was charged with 4.1 g diketone, 75 mL methanol and 1 mL 1 L methanolic sodium hydroxide prepared by the method described in Example 31. The suspension was stirred at room temperature. A homogeneous solution was obtained within a few minutes and a precipitate was observed after about 20 minutes. Stirring was continued for 70 minutes at room temperature and at the end the solid was filtered off and washed with methanol. The solid was dried in a steam cabinet to yield 3.6 g of 7-methylhydrogen 5β-cyano-11α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone .

1 H NMR (CDCl ₃ ) ppm 0.95 (s, 3H), 1.4 (s, 3H), 3.03 (d, 1H, J15), 3.69 (s, 3H), 4.1 (m, 1H).

13C NMR (CDCl ₃ ) ppm 14.6, 19.8, 22.6, 29.0, 31.0, 33.9, 35.17, 35.20, 36.3, 37.7, 38.0, 38.9, 40.8, 42.8, 43.1, 45.3, 45.7, 47.5, 52.0, 68.0, 95.0, 121.6 , 174.5, 176.4, 207.0.

Example 32B-2

7-Methylhydrogen 5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone Manufacturing.

To 2.0 g (4.88 mmol) of 9,11-epoxydiketone of formula 21 suspended in 30 mL of anhydrous methanol was added 0.34 mL (2.4 mmol) of triethylamine. The suspension was heated to reflux and after 4.5 h HPLC (Zorbax SB-C8 150 × 4.6 mm, 2 ml / min., Linear gradient over 15 min 35:65 A: B to 45:55 A: B, A = acetonitrile / Methanol 1: 1, B = water / 0.1% trifluoroacetic acid, detection at 210 nm), no raw material remained. The mixture was cooled and held at 25 degrees for about 16 hours. The obtained suspension was filtered to give 1.3 g of white solid 7-methylhydrogen 5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone was obtained. The filtrate was concentrated to a rotary evaporator to dryness and the remainder was titrated with 3-5 mL of methanol. Filtration gave an additional 260 mg of 7-methylhydrogen 5β-cyano-9,11α-epoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone. The yield was 74.3%.

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

Example 32C

5β-cyano-11-α, 17-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone Manufacture

The reaction flask was filled with 6.8 g of diketone (prepared as described in Example 31), 68 mL of acetonitrile, 6.0 g of sodium acetate and 60 mL of water. The mixture was warmed and stirred at reflux. After about 1.5 hours the reaction was nearly homogenized. After 3 hours of distillation of 50 mL of acetonitrile, 100 mL of water was added. The mixture was cooled and the precipitated solid (1.7 g) was removed by filtration. The filtrate (pH = 5.5) was treated with hydrochloric acid to lower the pH to about 4.5 and precipitate a solid. The solid was separated, washed with water and dried to give 4.5 g of 5β-cyano-11-α, 7-dihydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylic acid, γ-lactone .

¹ H NMR (DMSO) ppm 0.8 (s, 3H), 1.28 (s, 3H), 3.82 (m, 1H).

¹³ C NMR (DMSO) ppm 14.5, 19.5, 22.0, 28.6, 30.2, 33.0, 34.1, 34.4, 36.0, 37.5, 37.7, 38.5, 42.4, 42.6, 45.08, 45.14, 47.6, 94.6, 122.3, 176.08, 176.24, 207.5 .

Example 33

Scheme 1: Step 3A: Method C: Preparation of methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

A diketone (30 g), prepared according to the method described in Example 31, was charged to a clean, dry three-necked reaction flask equipped with a thermometer, Dean Stark trap and mechanical stirrer. Methanol (24 mL) was charged to the reactor at room temperature (22 ° C.) and the resulting slurry was stirred for 5 minutes. A 25% by weight solution of sodium methoxide in methanol (52.8 mL) was charged to the reactor and the mixture was stirred at room temperature for 10 minutes while the mixture turned to a light brown clear solution and some exotherm was observed (2-3 ° C.). The addition rate was adjusted so that pot temperature did not exceed 30 degreeC. Thereafter, the mixture was heated to reflux (about 67 ° C.) and held under reflux for 16 hours. The sample was then recovered and analyzed for conversion by HPLC. The reaction was continued under reflux until the remaining diketone was below 3% of the diketone charge. While filling reflux 4N HCl (120 mL) into the reaction port, quenched HCN was formed in the scrubber.

As a result, 90-95% of methanol solvent was distilled out of the reaction mixture at atmospheric pressure. During the distillation the head temperature changed to 67-75 ° C. and the distillate containing HCN was treated with preservative caustic and bleach. After removal of the methanol the reaction mixture was cooled to room temperature and a solid product began to precipitate when the mixture was cooled to within the range of 40-45 ° C. An aqueous solution optionally containing 5 wt% sodium bicarbonate (1200 mL) at 25 ° C. was placed in a cooled slurry and the resulting mixture was then cooled to 0 ° C. in about 1 hour. Sodium bicarbonate treatment was effective to remove residual unreacted diketone from the reaction mixture. The solid product methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate and γ-lactone were recovered by filtration and the filter cake was washed with water (100 mL). The slurry was stirred at 0 ° C. for 2 hours to complete precipitation and recrystallization. The product was dried under 26 "mercury vacuum at 80-90 ° C. to yield a weight. The water content after drying was less than 0.25% by weight and the controlled molar yield was about 77-80% by weight.

Example 34

Scheme 1: Step 3A: Method D: Preparation of methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylic acid, γ-lactone.

A diketone prepared as in Example 31 (1 eq.) Was reacted with sodium methoxide (4.8 eqs) in methanol solvent in the presence of zinc iodide (1 eq.). Finishing of the reaction product can be carried out in connection with the extraction process described herein or by a non-extraction process in which methylene chloride extraction, brine and caustic washing, and sodium sulfate drying steps are removed. Also in the non-extraction process, toluene was replaced with 5% by weight sodium bicarbonate solution. Methylhydrogen 11α, 17α-dihydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate and γ-lactone were isolated as products.

Example 35

Scheme 1: Step 3C: Method C: Preparation of methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

The hydroxyester (1.97 g) prepared as in Example 34 was combined with tetrahydrofuran (20 mL) and the resulting mixture was cooled to -70 ° C. Sulfuryl chloride (0.8 mL) was added and the mixture was stirred for 30 minutes before imidazole (1.3 g) was added. The reaction mixture was warmed to room temperature and stirred for an additional 2 hours. The mixture was then diluted with methylene chloride and extracted with water. The organic layer was concentrated to give the crude product methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate and γ-lactone (1.97 g). Small samples of the product were analyzed by HPLC. The analysis showed that the ratio 9,11-olefin: 11,12-olefin: 7,9-lactone was 75.5: 7.2: 17.3. Other conditions were as described above and when carried out at 0 ° C., the reaction resulted in a product having a distribution of 9,11-olefin: 11,12-olefin: 7,9-lactone 77.6: 6.7: 15.7. This process is combined with one step, which is the introduction and removal of leaving groups for the introduction of the 9,11-olefin structure of the esters, ie the reaction with sulfuryl chloride has the 11α-hydroxy group of the hydroxy ester of formula V This leads to ^{dehydrohalation} reaction, resulting in a structure of ^Δ9,11 . The formation of the esters is also effective without the use of a drying agent such as strong acid (such as formic acid) or acetic anhydride. The reflux step, which is an alternative to producing carbon monoxide, is also removed.

Example 36A

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

The hydroxyester (20 g) prepared as in Example 34, and methylene chloride (400 mL) were placed in a clean, dry three-necked round bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple. The resulting mixture was stirred at ambient temperature until a complete solution was obtained. The solution was cooled to 5 ° C. using an ice bath. Methanesulfonyl chloride (5 mL) was added to the CH ₂ Cl ₂ solution containing hydroxyester, and triethylamine (10.8 mL) was slowly added dropwise. The rate of addition was controlled so that the temperature of the reactants did not exceed 5 ° C. The reaction was very exothermic; Therefore cooling was necessary. The reaction mixture was stirred at about 5 ° C. for 1 hour. When the reaction was complete (HPLC and TLC analysis), the mixture was concentrated at about 0 ° C. under mercury vacuum 26 until the mixture became a thick slurry. The resulting slurry was diluted with CH ₂ Cl ₂ (160 mL) and the mixture was concentrated at about 0 ° C. under mercury vacuum 26 to give a concentrate. Purity of the concentrate (R ³ = H and -AA- and -BB- are both -CH ₂ -CH ₂ -mesylate products of formula IV, ie methylhydrogen 11α, 17α-dihydroxy-3-oxopregne 4-ene-7α, 21-dicarboxylate, methylhydrogen 17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicar at γ-lactone Carboxylate, γ-lactone) was 82% (% HPLC region). This material was used in the next reaction without separation.

Potassium formate (4.7 g), formic acid (16 mL), and acetic anhydride (8 mL, 0.084 mol) were placed in a clean, dry reactor equipped with a mechanical stirrer, ruffler, thermocouple, and heating mantle. The resulting solution was heated to 70 ° C. and stirred for about 4-8 hours. Since addition of acetic anhydride is exothermic and generates gas (CO), the rate of addition had to be adjusted to control temperature and gas evolution (pressure). The reaction time for preparing the deactivator was determined according to the water content in the reactants (potassium and potassium formate, each containing 3-5% water). The elimination reaction is sensitive to water content; If> 0.1% water (KF) is present, the level of impurity of 7,9-lactone will be increased. It is difficult to remove this by-product from the final product. If KF indicated <0.1% water, deactivator was transferred to a concentrate of mesylate (0.070 moles) prepared in the previous step. The resulting solution was heated to 95 ° C. and volatiles were removed by distillation to recover from Dean Strak trap. When generation of volatiles ceased, Dean Strak traps were replaced with chucks and the reaction mixture was heated at 95 ° C. for an additional hour. Upon completion (TLC and HPLC analysis; <0.1%, raw material), the contents were cooled to 50 ° C. and vacuum distillation (mercury 26/50 ° C.) was started. The mixture was concentrated to a thick slurry and then cooled to ambient temperature. The resulting slurry was diluted with ethyl acetate (137 mL), the solution was stirred for 15 minutes and diluted with water (137 mL). The layers were separated and the lower aqueous layer was reextracted with ethyl acetate (70 mL). The combined ethyl acetate solution was washed once with brine solution (120 mL) and twice with ice cold 1N NaOH solution (120 mL each). When the pH of the washed liquid was <8, the pH of the aqueous layer was measured and the organic layer was washed again. When the pH of the washed liquid was> 8, the ethyl acetate layer was washed once with brine solution (120 mL) and concentrated to rotary evaporation using a 50 ° C. water bath to form a dried product. The ester of the obtained solid product, ie, methyl hydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone, was 92 g (77 mol% yield). Weighed out).

Example 36B

Preparation of methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone.

25 g (53.12 mmol) of hydroxy ester methylhydrogen 11α, 17α-dihydroxy-3-oxo as in Example 34 in a clean dry 250 mL three-neck round bottom flask equipped with a mechanical stirrer, addition funnel and thermocouple. Pregan-4-ene-7α, 21-dicarboxylate, γ-lactone followed by 150 mL of methylene chloride (Burdick & Johnson) was added. The resulting mixture was stirred at ambient temperature until a small slurry was obtained. The solution was cooled to -5 ° C using an ice bath. Methanesulfonyl chloride (7.92 g, 69.06 mmol) (Aldrich) was added to a solution of methylene chloride containing hydroxyester, and triethylamine (7.53 g) was rapidly added dropwise. The rate of addition was adjusted so that the temperature of the reactants did not exceed 0 ° C. The reaction was very exothermic; Therefore cooling was necessary. The addition time was 35 minutes. The reaction mixture was stirred at about 0 ° C. for an additional 45 minutes. When the reaction was complete (when remaining hydroxyester was less than 1% in HPLC and TLC analysis), the mixture was concentrated by stripping about 100 mL to 125 mL of methylene chloride solvent at atmospheric pressure. The reactor temperature reached about 40 ° C. to 45 ° C. during the stripping process. If the reaction did not complete after an additional 45 minutes, additional 0.1 equivalents of methanesulfonyl chloride and additional 0.1 equivalents of triethylamine could be placed in the reactor and the reaction was confirmed to be complete. The resulting mixture contained the crude product methylhydrogen 17α-hydroxy-11α- (methylsulfonyl) oxy-3-oxopregan-4-ene-7α, 21-dicarboxylate and γ-lactone. This material was used in the next reaction without separation.

Anhydrous sodium acetate (8.7 g) (Mallinkrodt), crystalline acetic acid (42.5 mL) (Fisher) and acetic anhydride (0.5 mL) (Fisher) were cleaned and dried in a second 250 mL clean and dry machine equipped with a mechanical stirrer, ripper, thermocouple and heating mantle. Into the reactor. The resulting solution was heated to 90 ° C. and stirred for about 30 minutes. The addition of acetic anhydride was exothermic and the rate of addition had to be controlled to control both temperature and pressure. Acetic anhydride was added to reduce the water content of the solution to an acceptable level (up to about 0.1%). If KF indicates <0.1%, the acetic acid solution was transferred to the mesylate concentrate prepared as discussed in the first paragraph of this example. The temperature of the resulting mixture was about 55 ° C. to 60 ° C. after transfer. Gas evolution was reduced by using acetic acid and sodium acetate in place of formic acid and potassium formate as in Example 36A.

The mixture was heated to 135 ° C. and held at that temperature for about 60 to 90 minutes until the evolution of volatiles ceased. Volatile distilled from the mixture was recovered in a Dean Stark trap. Upon completion (TLC and HPLC analysis; <0.1% raw material) the heat source was removed. When the temperature of the mixture reached 80 ° C., 150 mL of water was slowly added to the mixture over 60 to 90 minutes. At the end of the water addition, the mixture was cooled to a temperature of about 35 ° C. to 45 ° C. and the slurry began to form. The mixture was further cooled to 15 ° C. and held at that temperature for about 30 to 60 minutes.

The mixture was filtered through a glass funnel. The filtrate was rinsed with 100 mL of water. The filtrate was then washed second with an additional 100 mL of water. The resulting filtrate was dried at 70 ° C. in vacuo to give 25.0 g of dry product ester, methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone was obtained. HPLC quantification showed 70% of the desired 9,11-olefin, 15% 11,12-olefin, and 5% 7,0-lactone.

This method is characterized by i) reducing the solvent volume, ii) reducing the number of separate operating steps required to prepare the esters from the hydroxyesters, iii) reducing the cleaning agents required, and iv) separating the final product. Replacing with precipitation and v) using formic acid instead of acetic acid has the beneficial consequences (compared to similar processes) of removing safety concerns in advance with regard to mixed anhydride formation and gas evolution.

Example 37A

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

The hydroxyester (100 g; 0.22 mol) prepared as in Example 34 was placed in a 2L three-necked round bottom flask equipped with a mechanical stirrer, addition funnel, and thermocouple. A rotary cooling bath was used with the thermostat. The flask was dried before the reaction due to the sensitivity of methanesulfonyl chloride to water.

Methylene chloride (1 L) was placed in a flask and hydroxyester was dissolved therein under stirring. The solution was cooled to 0 ° C. and methanesulfonyl chloride (25 mL; 0.32 mole) was added to the flask through an addition funnel. Triethylamine (50 mL; 0.59 mole) was added to the reactor through an addition funnel and the funnel was washed with additional methylene chloride (34 mL). The addition of triethylamine was very exothermic. The addition time was about 10 minutes under stirring and cooling. The charge mixture was cooled to 0 ° C. and left at that temperature under stirring for an additional 45 minutes where the top space of the flask turned red with nitrogen. Samples of the reaction mixture were then analyzed by thin layer chromatography and high performance liquid chromatography to confirm completion of the reaction. The mixture was then stirred for an additional 30 minutes at 0 ° C. and again checked for completeness. As a result of the analysis, the reaction was substantially completed at this point; Solvent methylene chloride was stripped under 26 "mercury vacuum at 0 ° C. Gas chromatography analysis of the distillate showed the presence of both methanesulfonyl chloride and triethylamine. Methylene chloride (800 mL) was then placed in the reactor. The resulting mixture was stirred for 5 minutes in the range 0-15 ° C. The solvent was again stripped at 0-5 ° C. under 26 ”mercury vacuum to give R ³ = H, -AA- and -BB- to -CH ₂ -CH _2. And a mesylate of formula IV wherein R ¹ is methoxy carbonyl. The purity of the product is about 90-95% area.

To prepare the remover, potassium formate (23.5 g; 0.28 mol) and acetic anhydride (40 mL) were mixed in a separate dry reactor. Formic acid and acetic anhydride were injected into the reactor and the temperature was kept below 40 ° C. during the addition of acetic anhydride. The remover mixture was heated to 70 ° C. to clean off moisture from the reaction system. This reaction was continued until the moisture content was less than 0.3% by weight as determined by Karl Fisher analysis. The degreasing agent solution was then transferred to a reactor containing the concentrated original mesylate solution prepared as described above. The resulting mixture was heated to a maximum temperature of 95 ° C. and volatile distillates were recovered until no further distillate was generated. Distillation stopped at about 90 ° C. After the distillation was completed, the reaction mixture was stirred for additional 2 hours at 95 ° C. and the reaction was confirmed by thin layer chromatography. After the reaction was completed, the reactor was cooled to 50 ° C. and formic acid and solvent were removed from the reaction mixture under 26 "mercury vacuum. The concentrate was cooled to room temperature and then ethyl acetate (688 mL) was injected and the mixture of ethyl acetate and concentrate The mixture was stirred for 15 minutes At this point, 12% brine solution (688 mL) was injected to help remove water soluble impurities from the organic phase The aqueous layer was transferred to another vessel filled with additional ethyl acetate (350 mL). This back extraction was carried out for 30 minutes after the phases were settled and the ethyl acetate layer was combined, saturated sodium chloride solution (600 mL) was added to the combined ethyl acetate layer and stirred for 30 minutes. The layer was removed and further sodium chloride (600 mL) washing was performed The organic phase was separated from the second written alcohol. The organic layer was washed with 1 N sodium hydroxide (600 mL) under stirring for 30 minutes The phases were placed for 30 minutes to remove the aqueous layer The pH of the aqueous layer was checked and found to be> 7. (600 mL). The organic phase was finally concentrated at 50 ° C. under 26 ”mercury vacuum and the product was recovered by filtration. The final product was a foamy brown solid upon drying. 95.4 g of the ester product methylhydrogen 17α-hydroxy-3-oxopregna-4,9 (11) -diene-7α, 21-dicar quantified as 68.8% by further drying at 45 ° C. under reduced pressure for 24 hours. Cylates and γ-lactones were obtained. The molar yield was corrected 74.4% for both raw hydroxyester and final ester.

Example 37B

7-methylhydrogen 17-methyl-3-oxo-18-norpregna-4,9 (11), 13-triene-7α, 21-dicarboxylate Manufacture

The reaction flask was charged with 5.5 g of mesylate prepared by the method of Example 23, 55 mL of 94.3% formic acid and 1.38 g of potassium formate. The mixture was heated and stirred at reflux (104 ° C.) for 2 hours. At the end of two hours, formic acid was distilled off under reduced pressure. The rest was dissolved in ethyl acetate and washed with 10% potassium carbonate (50 mL). Some of the water soluble recovered was yellow. Ethyl acetate was washed with 5% sodium hydroxide (50 mL). Some of the water solubles were combined, acidified with dilute hydrochloric acid, and the insolubles extracted from ethyl acetate were extracted. Ethyl acetate was distilled off under reduced pressure to yield 1.0 g of a residue, 7-methylhydrogen 17-methyl-3-oxo-18-norpregna-4,9 (11), 13-triene-7α, 21-. Dicarboxylate was obtained.

¹ H NMR (CDCl ₃ ) ppm 1.5 (s, 3H), 1.4 (s, 3H), 1.4 (s, 3H), 3.53 (s, 3H), 5.72 (m, 1H).

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

Example 37C

7-Methylhydrogen 5β-cyano-17-hydroxy-3-oxo-17α-pregan-11-ene-7α, 21-dicarboxylate, γ-lactone Manufacturing.

The reaction flask was charged with 5.5 g of mesylate prepared according to the method of Example 23, 55 mL of 94.3% formic acid and 1.38 g of potassium formate. The mixture was heated and stirred at reflux (104 ° C.) for 2 hours. At the end of two hours, formic acid was distilled off under reduced pressure. The rest was dissolved in ethyl acetate and washed with 10% potassium carbonate (50 mL). The recovered water soluble portion was yellow. Ethyl acetate was washed with 5% sodium hydroxide (50 mL). Ethyl acetate was distilled off under reduced pressure to give 3.7 g residue. A portion of 3.4 g of the residue was chromatographed on 267 g Merck silica gel (40-63 μ). The product was recovered by extraction technique of ethyl acetate and toluene 37:63 (v / v). After drying the product, 0.0698 g of residue, 7-methylhydrogen 5β-cyano-17-hydroxy-3-oxo-17α-pregan-11-ene-7α, 21-dicarboxylate, γ-lactone Got.

¹ H NMR (CDCl ₃ ) ppm 1.03 (s, 3H), 1.22 (s, 3H), 3.70 (s, 3H), 5.60 (d, 1H, J10), 5.98 (d, 1H, J10).

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

Example 37D

9α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, bis (γ-lactone) Separation of

9α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, bis (γ-lactone), is a byproduct of 11-mesylate removal. Pure samples were separated from the reaction mixture of Example 37A via preparative liquid chromatography followed by reverse phase preparative HPLC. In addition, 73 g residue was chromatographed on 2.41 g Merck silica gel (40-63μ) with gradient extraction of ethyl acetate and toluene (20:80, 30:70, 40:60, 60:40, v / v). . A rich mixture of enamines (10.5 g) and 7,9-lactone were obtained in 60:40 fractions. Purification process was taken by TLC on an EMF plate with 60:40 (v / v) ethyl acetate, toluene extract and visually confirmed by sulfuric acid, SWUV. A portion of the mixture (10.4 g) was further purified via reverse phase HPLC over Kromasil C8 (7μ) and 30:70 (v / v) MilliQ water and acetonitrile mobile phase. 7,9-lactone (2.27 g) was separated into crystals from the mobile phase.

MIR cm ⁻¹ 1762 (7,9-lactone and 17-lactone), 1677,1622 (3-keto-Δ ^4,5 )

¹ H NMR (CDCl ₃ ) ppm 1.00 (s, 3H), 1.4 (s, 3H), 2.05 (d, 1H), 2.78 (d, 1H), 5.87 (s, 1H).

¹³ C NMR (CDCl ₃ ) ppm 13.2, 19.0, 22.2, 23.2, 26.8, 28.8, 29.5, 30.8, 33.1, 34.4, 35.1, 42.5, 43.6, 43.9, 45.0, 45.3, 89.9, 94.7, 129.1, 161.5, 176.0, 176.4, 196.9.

Theoretically C, 71.85 and H, 7.34; C, 71.68 and H, shown as 7.30.

Example 37E

7-methylhydrogen 5-cyano-17-hydroxy-3-oxo-17α-pregan-11-ene-7α, 21-dicarboxylate, γ-lactone Separation of

Compound 7-Methylhydrogen 5-cyano-17-hydroxy-3-oxo-17α-pregan-11-ene-7α, 21-dicarboxylate, γ-lactone 11-mesyloxy (Example 24) The reaction mixture was separated after removal of and separated after a number of preparative liquid chromatography. This was part of a less polar impurity mass as seen by TLC on an EMF plate with 30:70 (v / v) ethylacetate and methylene chloride extraction system and visually confirmed by sulfuric acid, SMUV. In general, these less polar impurities were separated from raw enamines via preparative liquid chromatography. Specifically, 534 g of raw enamine solution using ethyl acetate and toluene gradient extraction technology (20:80, 30:70, 40:60, 60:40, v / v) was used for 534 g of Merck silica gel (40- Chromatography on 63μ). Less polar impurities were concentrated in the 30:70 fraction. 12.5 g pools of less polar impurities were recovered in this manner. The material was further chromatographed through 550 g of Merck silica gel (40-63 μ) using ethyl acetate and methylene chloride gradient extraction techniques (5:95, 10:90, 20:80, 30:70, v / v). It was. A rich portion of the 20:80 fraction gave 1.2 g of residue. Chromatography of 1.2 g residue through 53 g Merck silica gel (40-63 μ) using acetone and methylene chloride gradient (3:97, 6:94, 10:90, 15:85, v / v) yielded 0.27 g. Of 7-methylhydrogen 5-cyano-17-hydroxy-3-oxo-17α-pregan-11-ene-7α, 21-dicarboxylate, γ-lactone from a rich portion of the 10:90 fraction come.

MS M + 425, C ₂₅ H ₃₁ NO ₅ (425.52).

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

¹ H NMR (CDCl ₃ ) ppm 0.92 (s, 3H), 1.47 (s, 3H), 2.95 (m, 1H), 3.65 (s, 3H), 5.90 (m, 1H).

¹³ C NMR (CDCl ₃ ) ppm 14.0 (18 CH ₃ ), 23.5 (15 CH ₂ ), 27.0 (19 CH ₃ ), 37.8, 38.5 and 40.9 (7,8 and 14 CH), 52.0 (OCH ₃ ), 95.0 (17C), 121.5 (23CN), 123.5 (11CH), 135.3 (9C), 174.2 and 176.2 (22 and 24 C0), 206 (3CO).

Example 37F

7-Methylhydrogen 17-hydroxy-3-oxo-11α- (2,2,2-trifluoro-1-oxoethoxy) -17α-pregin-4-ene-7α, 21-dicarboxylate Manufacturing.

The hydroxyester (2.0 g, 4.8 mmols) prepared by the method of Example 34 was added to 40 mL of methylene chloride in a clean dry three-necked round bottom flask equipped with a mechanical stirrer. Triethylamine (0.61 g, 6.10 mmols) and trifluoroacetic anhydride (1.47 g, 7.0 mmols) were then added to the solution. The mixture was stirred at ambient temperature overnight. The mixture was diluted with an additional 40 mL of methylene chloride. The mixture was then washed successively with 40 mL water, 40 mL 1N HCl, and 40 mL 1N NaOH solution. The resulting solution was dried over magnesium sulfate and concentrated to dryness to afford 3.2 g of light brown solid, 7-methylhydrogen 17-hydroxy-3-oxo-11α- (2,2,2-trifluoro-1-oxo Ethoxy) -17α-pregin-4-ene-7α, 21-dicarboxylate was obtained.

The residue was further analyzed by chromatography and purified. HPLC conditions: column-water geometry C18 (150 mm × 4.6 mm id, 5-micron particle size); Column temperature--ambient temperature; Mobile phase--acetonitrile / water, 30/70 vol); Flow rate--1.0 mL / min; Injection volume—-20 microliters; Sample concentration--1.0 mg / mL; Detection--UV at 210 nm; Pressure-- 1500psi; And runtime--45 minutes). TLC conditions: Absorbent--Merck silica gel 60 F ₂₅₄ ; Solvent system-ethylacetate / toluene, 65/35 vol; Visualization technology--shortwaves; And application amount--100 micrograms.

Example 37G

7-Methylhydrogen 11α- (acetyloxy) -17-hydroxy-3-oxo-17α-pregin-4-ene-7, 21-dicarboxylate, γ-lactone Manufacturing.

Hydroxyester (2.86 g, 6.87 mmol) prepared by the method of Example 34 was added to 15 mL of methylene chloride in a clean, dry, 3-necked, round bottom flask equipped with a mechanical stirrer. Triethylamine (1.39 g, 13.7 mmol), dimethylaminopyridine (0.08 g, 0.6 mmol) and acetic anhydride (1.05 g, 10.3 mmol) were then added to the solution. The mixture was stirred overnight at ambient temperature.

The mixture was diluted with 150 mL of ethyl acetate and 25 mL of water. This ethyl acetate solution was washed with 25 mL of citric acid solution. The solution was dried over magnesium sulfate and concentrated to dryness to give 3.33 g of light brown solid 7-methylhydrogen 11α- (acetyloxy) -17-hydroxy-3-oxo-17α-pregin-4-ene-7, 21-. Dicarboxylate and γ-lactone were obtained.

The residue was further analyzed by chromatography and purified. HPLC conditions: column-water geometry C18 (150 mm × 4.6 mm id, 5-micron particle size); Column temperature--ambient temperature; Mobile phase--acetonitrile / water, 30/70 vol); Flow rate--1.0 mL / min; Injection volume—-20 microliters; Sample concentration--1.0 mg / mL; Detection--UV at 210 nm; Pressure-- 1500psi; And runtime--45 minutes). TLC conditions: Absorbent--Merck silica gel 60 F ₂₅₄ ; Solvent system--methylene chloride / methanol, 95/5 volumes; Visualization technology--shortwaves; And application amount--100 micrograms.

Example 37H

Scheme 1: Step 3C: Method F: 7-Methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone Manufacturing.

Potassium formate (1.5 g, 0.018 mole), formic acid (60 mL, 1.6 mole) and acetic anhydride (29.5 mL, 0.31 mole) were placed in a clean, dry 250 mL reactor equipped with a mechanical stirrer, twill, thermocouple and heating mantle. The solution was stirred at 70 ° C. for 4 hours and then the mesylate of Formula IV was cooled to ambient temperature to provide a remover useful for conversion to the product of this example.

The TFA / TFA anhydride remover performed was added to 70.0 g (0.142 mol) mesylate prepared by the method of Example 23. The resulting mixture was heated to 95 ° C.-105 ° C. for 2.5 hours and the conversion was periodically checked by TLC or HPLC. The residue obtained was cooled to 50 ° C., diluted with ice water (1.4 L) and stirred for 1 hour. The mixture was left overnight at ambient temperature. The layers were separated and the aqueous phase was re-extracted with ethyl acetate (75 mL). The ethyl acetate solution was then washed successively with water / brine mixture (70 mL), another water / brine mixture (60 mL), 1N sodium hydroxide (60 mL), and a third water / brine mixture (60 mL). Brin strength was 12% by weight. The ethyl acetate solution was dried over sodium sulfate, filtered, concentrated with a rotary evaporator, and dried to obtain 4.5 g of a desired product and a mixture of unknown impurities. The ratio of impurities / products by HPLC region is about 50/15 each. The main product from this reaction was impurities identified as 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate and γ-lactone.

The mixture was purified by column chromatography to give 1.9 g of analytically pure 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone was obtained.

The residue was further analyzed and purified by chromatography. HPLC conditions: column-water geometry C18 (150 mm × 4.6 mm id, 5-micron particle size); Column temperature--ambient temperature; Mobile phase--acetonitrile / water, 30/70 vol); Flow rate--1.0 mL / min; Injection volume—-20 microliters; Sample concentration--1.0 mg / mL; Detection--UV at 210 nm; Pressure-- 1500psi; And runtime--45 minutes). TLC conditions: Absorbent--Merck silica gel 60 F ₂₅₄ ; Solvent system--chloroform / methyl t-butyl ether / isopropanol, 70/28/2 vol; Visualization --50% by volume aqueous H ₂ SO ₄ / LWUV and 50 vol.% H ₂ SO ₄ / mold rib dinsan; And application amount--100 micrograms.

Example 37I

7-Methylhydrogen 17-hydroxy-3,11-dioxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone Manufacturing.

Jones is prepared by dissolving 6.7 g of chromic anhydride (CrO ₃ ) in 6 mL of concentrated sulfuric acid and carefully diluting the mixture with distilled water to form 50 mL. 1 mL of this formulation is sufficient to oxidize 2 mmol of secondary alcohol to ketones.

The hydroxyester (10.0 g, 24.0 mmol) prepared by the method of Example 34 was dissolved / stopped in 1200 mL acetone. To this mixture was added 8.992 mL of Jones and the combined mixture was stirred for 10 minutes. After treatment with water and extraction with methylene chloride, an aliquot of the reaction mixture was purified by HPLC (column: Beckman Ultrasphere ODS C18, 4.6 mm × 250 mm, 5 microns; solvent gradient: acetonitrile / water = 1.5 mL / min in 20 minutes). / 99 to 100/0; detector: UV 210 nm). The reaction was complete as evidenced by the lack of substantial amounts of raw materials in the reaction mixture. HPLC retention time for the raw material (hydroxyx) was 13.37 min and for product ketone 14.56 min.

The reaction was finished by adding 200 mL of water and 300 mL of methylene chloride. The organic layer was separated from the aqueous layer and washed again with 200 mL of water. The organic layer was separated from the aqueous layer and dried over magnesium sulfate. The solvent was distilled off to give 9.52 g of a off-white solid (95.6% raw yield) 7-methylhydrogen 17-hydroxy-3,11-dioxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone was provided.

Structural studies were based on mass spectrum (m / e 414), HNMR (DMF-d7) and CNMR (DMF-d7). In HNMR, there was no characteristic peak of 11-H (4.51 ppm, doublet, j = 5.8 Hz) seen in the hydroxyester. CNMR showed a peak expected at 11-keto carbon at 208.97 nm.

CNMR (400 MHz, DMF-d7) 208.97 (11-keto), 197.70 (3-keto), 176.00 (22-lactone), 173.34 (7-COOMe), 167.21 (C5), 125.33 (C4), 93.63 (C17) And other peaks in the region of 15-57 ppm.

Example 37J

Dimethyl 11α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone Manufacturing.

3.5 g (8.4 mmol) of hydroxyester solution prepared according to the method of Example 34 in 42 mL of methanol were mixed with 4 mL of methanolic 4N potassium hydroxide (8 mmols). The slurry was stirred at rt overnight and heated to reflux for 1 h. Methanol was evaporated in vacuo and the remainder was mixed with 50 mL of ethyl acetate. Ethyl acetate was evaporated in vacuo and the remainder was mixed with 50 mL of ethyl acetate. The dry solid was combined with 50 mL acetone and 2 mL methyl iodide (32.1 mmols). The mixture was stirred at rt for 18 h. During this time most of the solid was dissolved. The mixture was filtered and the filtrate was evaporated to dryness in vacuo. The residue was digested with ethyl acetate, the solids were removed by filtration and the solvents were removed under vacuum distillation. The residue was subjected to H ¹ NMR via dimethyl 11α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone and raw hydroxyester 78: It was determined that this was a 22 (v / v) mixture. This mixture is suitable for use as an HPLC marker without further purification.

¹ H NMR (CDCl ₃ ) indicated the following numbers: ppm 0.93 (s, 3H), 1.37 (s, 3H), 3.64 (s, 3H), 3.69 (s, 3H).

Example 37K

11α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone Manufacture

To 11.86 g (28.5 mmol) of hydroxyester prepared in the method of Example 34, 50 mL of methanol and 20 mL of 2.5M NaOH were added. After 25 minutes, HPLC (Zorbax SB-C8 150 × 4.6 mm, 2 ml / min., Linear gradient over 15 minutes 35:65 A: B to 45:55 A: B, A = acetonitrile / methanol 1: 1, B = water / 0.1% trifluoroacetic acid, detection at 210 nm), some of the raw ester remained unreacted and 10 mL of 10M NaOH was added. After 1.5 hours, traces of the ester remained unreacted as determined by HPLC. The reaction mixture was left at about 25 degrees for 64 hours.

The mixture was diluted with 100 mL of water and made strong acid with 20 mL of concentrated hydrochloric acid. The resulting viscous precipitate was stirred until the precipitate became a suspension. The solid was isolated by filtration, rearranged in methanol and filtered to yield 3.75 g of brown solid. This material was dissolved in 8 mL of hot DMF and the mixture was diluted with 40 mL of methanol. The acid was crystallized and separated by filtration to give 1.7 g of a fluffy white solid 11α, 17-dihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone Got it.

1 H-nmr (400 MHz, deuterodimethyl sulfoxide) d 0.80 (s, 3H), 1.25 (s, 3H), 1.2-2.7 (m, 20H), 3.8 (brs, 1H), 4.45 (m, 1H), 5.50 (s, 1 H). No carboxyl protons were observed due to the presence of HOD peak at 3.4 ppm.

Example 38

Scheme 1: Step 3C: Method G: Preparation of 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone .

Repeating the procedure of Example 37A, but eliminating a number of washing steps by treating the reaction solution with ion exchange resin, basic alumina or basic silica. Treatment conditions with basic alumina or basic silica are shown in Table 38. Each of these treatments is an example for producing 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone It has been found to be effective in removing impurities without a large number of 44 cleaning agents.

  Element Set point Purpose of the experiment  Key result  Basic alumina   2g / 125g products Treat the reaction mixture with basic alumina to remove Et ₃ N.HCl salt, remove 1N NaOH and wash 1N HCl  Yield 93%  Basic silica   2g / 125g products Treatment of the reaction mixture with cheaper basic silica removes Et ₃ N.HCl salt, removes 1N NaOH, and washes 1N HCl  Yield 95%

Example 39

Scheme 1: Step 3C: Method H: Preparation of 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone .

Potassium acetate (4 g) and trifluoroacetic acid (42.4 mL) were mixed in a 100 mL reactor. Trifluoroacetic anhydride (9.5 mL) was added to the mixture at a controlled rate to maintain the temperature below 30 ° C. during the addition. The solution was heated to 30 ° C. for 30 minutes to provide a remover useful for converting the mesylate of Formula IV to the ester of Formula II.

A preformed TFA / TFA anhydride remover was added to the mesylate solution of Formula IV prepared as in Example 37A earlier. The resulting mixture was heated at 40 ° C. for 4½ hours and the conversion was checked periodically by TLC or HPLC. When the reaction was complete, the mixture was transferred to a 1-neck flask and concentrated to dryness at room temperature (22 ° C.). After addition of the water / brine mixture (137 mL), ethyl acetate (137 mL) was added to the mixture to obtain a complete solid lysate and the mixture of the two phases obtained was stirred for 10 minutes. The phases were allowed to separate for 20 minutes. The brine strength was 24% by weight. The aqueous phase was contacted with an added amount of ethyl acetate (68 mL) and the mixture of the two phases prepared was left for 15 minutes for phase separation and stirred for 10 minutes. The ethyl acetate layers were combined from the two extracts and washed with 24 wt% brine (120 mL), an aliquot of another 24 wt% brine (60 mL), 1N hydrogen peroxide solution (150 mL) and another portion of brine (60 mL). After each aqueous phase addition, the mixture was stirred for 10 minutes and allowed to stand for 15 minutes for separation. The resulting solution was concentrated under reduced pressure at 45 ° C. using a water aspirator to obtain a dried product. The solid product (8.09 g) was analyzed by HPLC and 83.4 area% of ester 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicar Carboxylate, γ-lactone; 11,12-olefins of 2.45 region%; 1.5% 7,9-lactone; And 1.1% unreacted mesylate.

Example 40

Scheme 1: Step 3C: Method I: Preparation of 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone .

Mesylate (1.0 g), isopropenyl acetate (10 g) and p-toluenesulfonic acid (5 mg) having a structure prepared per Example 23 were placed in a 50 ml flask and heated to 90 ° C while stirring. After 5 hours, the mixture was cooled to 25 ° C. and concentrated in vacuo at 10 mm of mercury. The residue was dissolved in CH ₂ Cl ₂ (20 ml) and washed with 5% aqueous NaHCO ₃ . The CH ₂ Cl ₂ layer was concentrated in vacuo to yield 1.47 g of tan oil. This material was recrystallized from CH ₂ Cl ₂ / Et ₂ O to obtain 0.50 g of enol acetate of formula IV (Z).

This material was added to a mixture of sodium acetate (0.12 g) and acetic acid (2.0 ml) previously heated to 100 ° C. with stirring. After 60 minutes the mixture was cooled to 25 ° C. and diluted with CH ₂ Cl ₂ (20 ml). The solution was washed with water (20 ml) and dried over MgSO ₄ . The drying agent was removed by filtration and the filtrate was concentrated in vacuo to give 0.4 g of the desired 9,11-olefin, 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene. -7,21-dicarboxylate and γ-lactone were obtained. The product had a 7,9-lactone impurity of less than 2%.

Example 41-Heat Removal of Mesyl Acid in DMSO

Scheme 1: Step 3C: Method J: Preparation of 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21-dicarboxylate, γ-lactone .

A mixture of 2 g mesylic acid and 5 ml DMSO in the flask was heated at 80 ° C. for 22.4 h. HPLC analysis of the reaction mixture showed that no raw material was detected. Water (10 ml) was added to the reaction and the precipitate was extracted three times with methylene chloride. The combined methylene chloride layers were washed with water, dried over magnesium sulfate and concentrated to give ester 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7,21- Dicarboxylate and γ-lactone were obtained.

Example 42

Scheme 1: Step 3D: Method B: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

In a 50 mL pear-shaped flask, an ester of formula IIA (74.4% quantification of 1,07 g), trichloroacetamide (0.32 g), solid dipotassium hydrogen phosphate (0.70 g) and methylene chloride (15.0 mL) Mix with stirring. Hydrogen peroxide (30% by weight; 5.0 mL) was added by pipette over 1 minute. The resulting mixture was stirred for 6 hours at room temperature where HPLC analysis indicated that the ratio of epoxymexrenone to esters in the reaction mixture was about 1: 1. Additional trichloroacetamide (0.32 g) was added to the reaction mixture and the reaction was continued with stirring for 8 hours more after the time showed that the ratio of the remaining ester was lowered to 10%. Additional trichloroacetamide (0.08 g) was added at the point where only 5% of unreacted ester remained for the epoxymexrenone in the mixture and the reaction mixture was allowed to stand overnight.

Example 43

Scheme 1: Step 3D: Method C: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

An ester of formula IIA (5.4 g, quantitative 74.4% ester) was placed in a 100 mL reactor. Both trichloroacetamide (4.9 g) and dipotassium hydrogen phosphate (3.5 g) were added to the ester in solid form, followed by methylene chloride (50 mL). The mixture was cooled to 15 ° C. and 30% hydrogen peroxide (25 g) was added over 10 minutes. The reaction mixture was brought to 20 ° C. and stirred at that temperature for 6 hours, at which point conversion was checked by HPLC. It was determined that the remaining ester was less than 1% by weight.

The reaction mixture was poured into water (100 mL), the phases were separated, and the methylene chloride layer was removed. Sodium hydroxide (0.5 N; 50 mL) was added to the methylene chloride layer. After 20 minutes to allow the phases to separate, the phases were separated and the organic phase was washed with saturated brine (50 mL) and HCl (0.5N; 50 mL) was added to the methylene chloride layer. The methylene chloride layer was dried over anhydrous magnesium sulfate and the solvent was removed. A white solid (5.7 g) was obtained. The aqueous sodium hydroxide layer was acidified and extracted to finish the extract to obtain 0.2 g of additional product. The yield of epoxymexrenone was 90.2%.

Example 44

Scheme 1: Step 3D: Method D: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

The ester of formula (IAA) was converted to epoxymexrenone as described in Example 43 with the following differences: The initial charge was ester (5.4 g quantitative 74.4% ester), trichloroacet Amide (3.3 g), and dipotassium hydrogen phosphate (3.5 g). Hydrogen peroxide solution (12.5 mL) was added. The reaction was carried out overnight at 20 ° C. after HPLC showed 90% conversion of the esters to the epoxymexrenone. Additional trichloroacetamide (3.3 g) and 30% hydrogen peroxide (5.0 mL) were added and the reaction was carried out for an additional 6 hours when the remaining ester was only 2% based on the ester charge. After finishing as described in Example 43, 5.71 g of epoxymexrenone was obtained.

Example 45

Scheme 1: Step 3D: Method E: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

Esters of formula (IA) were converted to epoxymexrenone as described generally in Example 43. In the reaction of the present example, the ester charge was 5.4 g (quantity 74.4%), the trichloroacetamide charge was 4.9 g, the hydrogen peroxide charge was 25 g, and the dipotassium hydrogen phosphate charge was 3.5 g. The reaction was run at 20 ° C. for 18 hours. Remaining ester was less than 2%. After finishing, 5.71 g of epoxymexrenone was obtained.

Example 46

Scheme 1: Step 3D: Method F: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

The ester of formula (IA) was converted to epoxymexrenone as described in Example 43 except that the reaction temperature was 28 ° C. Materials charged to the reactor included esters (2.7 g), trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g), hydrogen peroxide (17.0 g) and methylene chloride (50 mL). After 4 hours of reaction, the unreacted ester was only 2% based on the ester charge. After the finish described in Example 43, 3.0 g of epoxymexrenone was obtained.

Example 47-1

Scheme 1: Step 3D: Method G: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

An ester of formula (IAA) (40.4 g, quantitative 68.4% ester) was charged into a 1000 mL jacketed reactor and dissolved in 175 mL of methylene chloride. The solution was stirred while adding trichloroacetamide (22.3 g) and dipotassium hydrogen phosphate (6.0 g) in solid form. The mixture was stirred at 400 RPM and the temperature was adjusted to 27 ° C. with a thermostat to control the liquid rotating through the reactor jacket. Hydrogen peroxide (72.8 mL in 30% quantum) was added over 3-5 minutes. After addition of hydrogen peroxide, the mixture was stirred at 400 RPM and 27 ° C. HPLC quantification showed that the reaction was 99% complete within 5 hours. At the end of 6 hours, 72.8 mL of water was added. Aqueous hydrogen peroxide was separated and back extracted once with 50 mL of methylene chloride. The combined methylene chloride was washed with 6% sodium sulfite (62.3 mL) to destroy any contained peroxides. The methylene chloride removal started with atmospheric distillation and ended under vacuum. A yellowish residue (48.7 g, 55.4% quantification) was obtained. This corresponds to 94.8% quantification in terms of molarity.

A portion of the residue (47.8 g) was combined with 498 mL of ethanol 3A (95% ethanol modified with 5% methanol). The mixture was heated to reflux and 249 mL of distillate was removed at atmospheric pressure. The mixture was cooled to 25 ° C. and filtered. Ethanol 3A rinse (53 mL) was used to aid transfer. The dried solid weighed 27.6 g (87.0% quantification), which corresponds to 91% recovery. A portion of the solid (27.0 g) was dissolved in 292 mL of methyl ethyl ketone at reflux. The hot solution was filtered through a solka floc pad (powder cellulose) with another 48.6 mL of methylethylketone used to aid transfer. Some 146 mL of methyl ethyl ketone was removed via atmospheric distillation. The solution was cooled to 50 ° C. and stirred for one hour to crystallize the product. After one hour the mixture was cooled to 25 ° C. Stirring was continued for one hour and filtered with 48.6 mL of methylethylketone used as rinse water. The solid was dried to yield 20.5 g of a yield showing 87.2% recrystallization recovery. Reactant yield and ethanol, methyl ethyl ketone recovery were combined in a total of 75%.

The methylethylketone mother liquor is suitable for regeneration with methylene chloride coming in in the following reaction. The combined methylene chloride and methylethylketone mixture was evaporated by atmospheric distillation and vacuum distillation to form a dry matter. The residue was combined with 19 volumes of ethanol 3A based on epoxymexrenone content. After cooling to 25 ° C. the solid was filtered off and dried. The dry solid was dissolved in 12 volumes of methyl ethyl ketone at reflux. The hot solution was filtered through a solka floc pad with 2 volumes of methylethylketone added as a rinse. The filtrate was concentrated to 6 volumes of methyl ethyl ketone by atmospheric distillation. The solution was cooled to 50 ° C. and stirred for one hour to crystallize the product. After one hour the mixture was cooled to 25 ° C. Stirring was continued for one hour and the solid was filtered through two volumes of methylethylketone used as rinse water. The solid was dried to give a dose. Injection of methyl ethyl ketone mother liquor raised the total yield to 80-85%. This method is particularly suitable for large scale because it maximizes runoff and minimizes detergent volume and waste.

Example 47A

 Scheme 1: Step 3D: Method H: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

An ester of formula (IA) (17 g, quantitative 72% ester) was added to trichloroacetamide (14.9 g) under slow stirring and then dissolved in methylene chloride (150 mL). The temperature of the mixture was adjusted to 25 ° C. and a dipotassium hydrogen phosphate (10.6 g) solution in water (10.6 mL) was added to the ester substrate solution with 400 rpm stirring. Hydrogen peroxide (30 wt% solution; 69.4 mL) was added to the substrate / phosphate / trichloroacetamide mixture over 3-5 minutes. No exotherm or oxygen evolution was observed. The reaction mixture was stirred at 400 rpm and 25 ° C. for 18.5 hours. No oxygen evolution was observed throughout the reaction but hydrogen peroxide consumption analysis indicated that some oxygen was formed during the reaction. The reaction mixture was diluted with water (69.4 mL) and the mixture was stirred at about 250 rpm for 15 minutes. No temperature control is necessary for this operation and essentially performed at room temperature (any temperature within the range of 5-25 ° C. is acceptable). The aqueous and organic layers were allowed to separate and the lower methylene chloride layer was removed.

The aqueous layer was back extracted with methylene chloride (69.4 mL) for 15 minutes under stirring at 250 rpm. The layers were allowed to separate and the lower methylene chloride layer was removed. The aqueous layer (177 g; pH = 7) was allowed to determine the amount of hydrogen peroxide. The result (12.2%) indicated that 0.0434 mole of hydrogen peroxide was consumed during the olefin reaction of 0.0307. Excess hydrogen peroxide consumption was a measure of oxygen evolution in the reaction. Back extraction with a small volume of methylene chloride was sufficient to confirm that there was no loss of epoxymexrenone in the aqueous layer. This result was confirmed by the application of the second large amount of methylene chloride extraction in which only trichloroacetamide was recovered.

The combined methylene chloride solution from the extract described above was combined and washed with 3% by weight sodium sulfite solution (122 mL) at about 250 rpm for at least 15 minutes. A negative starch iodide test (KI paper; no color; purple coloration in the positive test indicates the presence of peroxides) was observed at the end of the agitation.

The aqueous and organic layers were allowed to separate and the lower methylene chloride layer was removed. The aqueous layer (pH = 6) was discarded. Note that the addition of sulfurous acid solution is slightly exothermic, so such addition should be carried out under temperature control.

The methylene chloride phase was washed with 0.5 N sodium hydroxide (61 mL) for 45 minutes at a temperature in the range of 15-25 ° C. (pH = 12-13) at about 250 rpm. Impurities derived from trichloroacetamide were removed in this process. Following the oxidation of the alkaline aqueous fractions, the extraction of methylene chloride confirmed that very few epoxymexrenones were lost in this operation.

The methylene chloride phase was washed once with 0.1 N hydrochloric acid (61 mL) for 15 minutes at a temperature in the range of 15-25 ° C. at about 250 rpm. The layers were allowed to separate and the lower methylene chloride layer was removed and washed again with 10% by weight aqueous sodium chloride (61 mL) for 15 minutes at a temperature in the range of 15-25 ° C. at about 250 rpm. The layers were separated again and the organic layer was removed. The organic layer was filtered through a solka floc pad and then evaporated under reduced pressure to form a dry matter. Drying was completed at a bath temperature of 65 ° C. A white off-white solid (17.95 g) was obtained for HPLC quantitation. Epoxymexrenone quantification was 66.05%. The conversion molar yield of the reaction was 93/1%.

The product was dissolved in hot methylethylketone (189 mL) and the resulting solution was distilled at atmospheric pressure until 95 mL of ketone solvent was removed. The temperature was lowered to 50 ° C. to allow the product to crystallize. The temperature was lowered back to 20-25 ° C. and stirring continued for 2 hours. The solid was filtered, rinsed with MEK (24 mL) and dried to yield 9.98 g of an amount that contained 93.63% epoxymexrenone according to HPLC quantification. This product was redissolved in hot MEK (106 mL) and the hot solution was filtered through a 10 micron line filter under pressure. Another 18 mL of MEK was applied as a rinse to distill the filtered MEK solution at atmospheric pressure until 53 mL of solvent was removed. The temperature was lowered to 20-25 ° C. and stirring was allowed to stand at that temperature for another two hours. The solid product was filtered off and rinsed with MEK (18 mL). The solid product was dried to give an 8.32 g amount containing 99.6% epoxymexrenone per quantitative HPLC determination. Final loss on drying was less than 1.0%. With respect to the reaction and finish of this example, the total yield of epoxymexrenone is 65.8%. This total yield reflected 93% reaction yield, 78.9% initial crystallization recovery, and 89.5% recrystallization recovery.

Example 47B

7-Methylhydrogen 11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone Manufacturing.

Δ ^11,12 olefins of the esters are by-products of 11-mesylate removal. Pure samples were separated from the reaction mixture prepared by the method of Example 37A via repeated preparative liquid chromatography. In addition, 73 g residue (prepared according to the method of Example 37A) was extracted with ethyl acetate and toluene gradient extraction technique (20:80, 30:70, 40:60, 60:40, v / v) of 2.41 kg of Merck silica gel. Chromatography on (40-63μ). Some of the rich Δ ^11,12 olefins were combined from the selected 30:70 fractions. Using ethylacetate / toluene 60:40 (v / v) with sulfuric acid SWUV identification, TLC on the EMF plate served as a guide in selecting the appropriate fraction. After removal of the solvent, 7.9 g of the original ^Δ11,12 olefin (80 area% in HPLC) was purified by ethyl acetate / methyl chloride gradient technique (10:90, 20:80, 35:65, v / v) to 531 g of Chromatography on Merck silica gel (40-63μ). Pure 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,11-diene-7α, 21-dicarboxylate, γ-lactone (3.72 g) was obtained from the selected 20:80 fraction. Selection of fractions is based on TLC generation as in the previous situation.

MIR cm- ¹ 1767 (lactone), 1727 (ester), 1668 and 1616 (3-keto-Δ ^4,5 ).

¹ H NMR (CDCl ₃ ) ppm 1.05 (s, 3H), 1.15 (s, 3H), 3.66 (s, 3H), 5.58 (dd, 1H), 5.80 (s, 1H), 5.88 (dd, 1H).

¹³ C NMR (CDCl ₃ ) ppm 17.41, 18.58, 21.73, 28.61, 32.28, 33.63, 34.91, 35.64, 35.90, 38.79, 42.07, 44.12, 48.99, 49.18, 51.52, 93.81, 126.43, 126.69, 133.76, 166.24, 172.172 176.64, 198.56.

1.6 g (3.9 mmols) of 7-methylhydrogen 17-hydroxy-3-oxo-17α-pregna-4,11-diene-7α, 21-dicarboxylate, γ-lactone solution was added to 16 mL of methylene chloride. 2.2 mL of trichloroacetonitrile (22.4 mmols) and 0.75 g of dipotassium phosphate (4.3 mmols) were mixed. The mixture was stirred and combined with 6.7 mL of 30% hydrogen peroxide (66 mmols). Stirring was continued at 25 ° C. for 45 hours. At the end of this time, 28 mL of methylene chloride and 39 mL of water were added. The organic portion was separated and washed successively with a) 74 mL of 3% sodium sulfite, b) 62 mL of 1N sodium hydroxide, c) 74 mL of 1N hydrochloric acid, and d) 31 mL of 10% brine. The organic portion was separated again, dried over magnesium sulfate and distilled under vacuum to dryness. The 1.25g residue was chromatographed on 138.2g Merck silica gel (40-63μ) using methyl-t-butylether and toluene gradient systems (40:60, 60:40, 75:25, v / v). Suitable ratios of the 60:40 and 75:25 fractions were combined after TLC generation to yield 0.66 g of pure 7-methylhydrogen 11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α , 21-dicarboxylate and γ-lactone were obtained. The TLC system used 75:25, (v / v) methyl-t-butylether and toluene extraction techniques with EMF plates with sulfuric acid and SWUV for visual identification.

¹ H NMR (CDCl ₃ ) ppm 1.09 (s, 3H), 1.30 (s, 3H), 3.05 (for AB ^11,12 2H), 3.67 (s, 3H), 5.80 (s, 1H).

¹³ C NMR (CDCl ₃ ) ppm 14.2, 18.0, 21.2, 28.8, 31.9, 33.5, 34.6, 34.7, 35.1, 35.5, 37.4, 38.3, 41.8, 46.0, 47.2, 50.4, 51.7, 56.7, 94.0, 126.7, 165.2, 172.5, 176.7, 198.1.

In theory: C, 69.54 and H, 7.30; Represented by C, 69.29 and H, 7.17.

Example 47C

7-Methylhydrogen 4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone Separation.

The raw epoxymexrenone (157 g) prepared by the method of Example 26 from 200 g of ester was subjected to chromatography on 4.4 kg of Merck silica gel (40-63 μ). A portion of 88.1 g was recovered by acetonitrile and toluene 10:90 (v / v) extraction techniques. The separated solid was dissolved in 880 mL hot methylethylketone and filtered through solka floc pad. Another 88 mL of methylethylketone was applied as a rinse. The filtrate was concentrated through removal of 643 mL of solvent and the mixture was cooled to room temperature. The solid was filtered off and rinsed with methylethylketone. After drying, 60.2 g of epoxymexrenone was obtained, which was quantified as 96.8% by HPLC. The filtrate was concentrated to dryness under reduced pressure. 9.3 g residue was recrystallized from 99 mL methyl ethyl ketone to give 2.4 g dry solid. Some 400 mg of the solid was subjected to reverse phase preparative HPLC on a YMC ODS AQ column. Pure 7-Methylhydrogen 4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone (103 mg) was acetonitrile (24 %), Methanol (4%) and water (72%) extraction techniques.

¹ H NMR (CDCl ₃ ) ppm 0.98 (s, 3H), 1.32 (s, 3H), 2.89 (m, 1H), 3.07 (s, d, 2H), 3.73 (s, 3H).

MS, M + 430, calculated by C ₂₄ H ₃₉ O ₇ (430.50).

Example 47D

7-Methylhydrogen 17-hydroxy-3,12-dioxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone Separation.

The methyl ethyl ketone mother liquor obtained by the method of Example 26 was distilled off under reduced pressure and dried. A portion of the remaining 4.4 g was chromatographed on 58.4 g BTR Zorbax LP (40 μ). Extraction of a gradient of methylethylketone and methylene chloride (25:75 to 50:50, v / v) resulted in 1.38 g of material. A portion of 1.3 g of this material was further purified via reverse phase preparative HPLC using acetonitrile (30%), methanol (5%) and water (65%) and YMC ODS AQ column (10μ) as mobile phase. The product was obtained from the fractions enriched through methylene chloride extraction. Methylene chloride was evaporated to dryness and the 175 mg residue was reconstituted via reverse phase preparative HPLC using acetonitrile (24%), methanol (4%) and water (72%) and a YMC ODS AQ column as mobile phase. Abundant fractions of methylene chloride extract were purified to 30.6 mg pure 7-methylhydrogen 17-hydroxy-3,12-dioxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone was brought.

¹ H NMR (CDCl ₃ ) ppm 1.17 (s, 3H), 1.49 (s, 3H), 3.13 (m, 1H), 3.62 (s, 3H), 5.77 (s, 1H), 5.96 (s, 1H).

¹³ C NMR (CDCl ₃ ) ppm 13.1, 21.0, 28.0, 29.4, 33.1, 33.4, 33.9, 35.5, 36.7, 40.3, 41.5, 43.0, 43.4, 52.0, 55.0, 91.0, 123.7, 126.7, 163.2, 167.9, 171.8, 176.8, 197.4, 201.0.

Example 47E

9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, dihydrate, dipotassium salt Manufacturing.

A suspension comprising 2.0 g (4.8 mmol) of epoxymexrenone, 10 mL of water, 3 mL of dioxane and 9.3 mL of 1.04N water soluble potassium hydroxide (9.7 mmol) prepared by the method of Example 43 was prepared. The mixture was stirred at 25 ° C. for 3 hours. A yellow, homogeneous solution was formed for the first two hours. The temperature was raised to 70 ° C. and stirring continued for an additional 3 hours. The solvent was removed via vacuum distillation and the remainder was purified by reverse phase chromatography on 90 g of C18 silica gel using water as extractant. The desired fractions were taken by TLC on an EMF plate using methylene chloride, methanol (7: 3) as extractant and visually confirmed using SWUV and combined. The combined fractions were concentrated in vacuo to dryness and the residue was allowed to be reversed-phase purified and repeated as described previously. The desired fractions were concentrated to dryness under reduced pressure and the residue was dissolved in ethanol. Ethyl acetate was added to the cloud point and heptane was added to complete the precipitation. 0.55 g of the product, 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, dihydrate, dipotassium salt were isolated as a yellow solid. . Carbon analysis was associated with the hydroxyl structure C ₂₃ H ₂₈ O ₇ K ₂ · 1.75 H ₂ O: theoretical C, 52.50 versus 55.85 for the anhydride form; C, shown as 52.49. As an extractant, Tf on the EMF plate with methylene chloride, methanol, and water (6: 3: 0.5, v / v) was visually confirmed through SWUV. An R _f of 0.29 was observed.

Example 47F

9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, dihydrate, disodium salt Manufacturing.

About 5 mg (0.01 mol) of epoxymexrenone prepared by the method of Example 43 was suspended in about 200 μL of methanol in a 4 mL vial and diluted with about 200 μL of 2.5N NaOH. The resulting mixture was a yellow homogeneous. The mixture was heated to 70 ° C. in an oil bath. After 10 minutes, a 1 μm sample was taken from the mixture using HPLC (Zorbax SB-C8 150 × 4.6nn, 2 mL, showing two substances at 4.86 and 2.93 min retention times, which matched the hydroxyl (open lactone) and open lactone 7-carboxylic acids, respectively. / Min, gradient = 35: 65 (v / v) A: B, A = acetonitrile / methanol (1: 1), B = water / 0.1% trifluoroacetic acid, detection at 210 nm). After 30 minutes the second (0.05 mL) sample was removed and oxidized with about 0.05 mL of 3N HCl and then neutralized with about 0.5 mL of sodium bicarbonate. Such HPLC analysis showed the expected ring-closed steroid with retention times of 6.59 and 10.71 minutes. 7-Methylhydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, γ-lactone for the corresponding 7-carboxylic acid ( 10.71 minutes) was 7:89.

Selective hydrolysis of lactones was possible under mild conditions. A second 4 mL vial was prepared as above but not heated. The mixture was sonicated for 5 minutes. 0.05 mL of sample was diluted in 0.5 mL of 1: 1 (v / v) methanol / acetonitrile mixture and analyzed by HPLC without prior oxidation. The open lactone carboxylic acid 7-ester obtained had a retention time of 4.85 minutes as observed above and was not contaminated with 7-carboxylic acid.

Example 47G

7-methylhydrogen 9α, 11β, 17-trihydroxy-3-oxo-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone And 7-Methylhydrogen 12α, 17-dihydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone Separation of

7-Methylhydrogen 9α, 11β, 17-trihydroxy-3-oxo-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone and 7-methylhydrogen 12α, 17-dihydrate Roxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone as described in Example 26 (trichloroacetonitrile protocol) epoxy of the ester 2-butane mother liquor recovered by firing was separated after liquid chromatography. In addition, the first crystallization was carried out using 2-butanone as directed. However, recrystallization used 2-butanone (10 vol / g) instead of acetone. 2.8 g residue was obtained in this way and purified via reverse phase preparative HPLC. Chromasil C8 (10μ) was used as the stationary phase with a mobile phase consisting of milliQ water and acetonitrile in a ratio of 70:30 (v / v). Crystallization was observed in one of the rich fractions. The solid (46.7 mg) was isolated and identified as 7-methylhydrogen 9α, 11β, 17-trihydroxy-3-oxo-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone . The mother liquor was evaporated to dryness under reduced pressure, and the remaining (123 mg) was purified by 7-methylhydrogen 12α, 17-dihydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxyl. It was confirmed that the rate, γ-lactone.

7-Methylhydrogen 9α, 11β, 17-trihydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone:

MS M + 432 calculated by C ₂₄ H ₃₂ O ₅ (432.51).

¹ H NMR (CDCl ₃ ) ppm 1.23 (s, 3H), 1.54 (s, 3H), 3.00 (m, 1H), 3.14 (m, 1H), 3.74 (m, 1H), 5.14 (s, 1H, slowly Interchangeable), 5.79 (s, 1H).

¹³ C NMR (CDCl ₃ ) ppm 16.8, 22.7, 24.8, 29.0, 29.3, 32.1, 34.1, 34.7, 35.2, 35.7, 36.8, 40.7, 43.0, 45.0, 45.9, 52.9, 72.8, 77.4, 95.9, 127.4, 163.7, 176.7, 177.3, 199.4.

7-Methylhydrogen 12α, 17-dihydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone:

MS M + 441 calculated by C ₂₄ H ₃₀ O ₆ (414.50).

¹ H NMR (CDCl ₃ ) ppm 0.87 (s, 1H), 1.40 (s, 1H), 3.05 (m, 1H), 3.63 (s, 3H), 3.99 (m, 1H), 5.72 (s, 1H), 5.96 (m, 1 H).

¹³ C NMR (CDCl ₃ ) ppm 14.8, 24.0, 26.1, 29.7, 33.6, 33.8, 34.0, 36.3, 37.0, 37.4, 40.7, 40.9, 43.8, 48.1, 51.9, 69.1, 95.5, 122.7, 126.3, 145.9, 164.5, 173.2, 177.6, 198.2.

Example 47H

7-methylhydrogen 9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone And 7-Methylhydrogen 6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone Manufacturing.

7-Methylhydrogen 9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregin-4-ene-7α, 21-dicarboxylate, RMWeier, which includes γ-lactone by reference; and Prepared according to the method of LMHofmann (J. Med Chem 1977,1304). 148 g (357 mmols) of 7-methylhydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate prepared by the method of Example 43, γ-lactone was combined with 311 mL of Absolut ethanol and 155 mL (932 mmols) of triethylortho formic acid. The slurry was stirred at room temperature and 10.4 g (54.7 mmols) of toluenesulfonic acid (monohydroxide) was added as catalyst. Stirring was continued for 30 minutes and the reaction was quenched with addition of 41.4 g (505 mmols) of powdered sodium acetate and 20.7 mL (256 mmols) of pyridine. The solid (70.2 g) was removed by filtration and the filtrate was concentrated in vacuo to yield a dry matter. The remaining 300mL of ethyl acetate and 9.8g of solid were removed by filtration.

The filtrate was concentrated to dryness and the residue was digested with 100 mL of methanol containing 2 mL of pyridine. 29.7 g of solids were removed by filtration. Additional precipitate was observed in the filtrate. Thus, the filtrate was refiltered to remove additional 21.9 g solids. The filtrate was concentrated to dryness and the residue was digested with 50 mL methanol containing 1 mL pyridine. 33.8 g of solid was separated by filtration. Quantitative HPLC showed that the last part of this solid was sufficiently pure (90 area percent of 7-methylhydrogen 9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregin-4- En-7α, 21-dicarboxylate, γ-lactone).

7-Methylhydrogen 9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone:

¹ H NMR (CDCl ₃ ) ppm 1.02 (s, 3H), 1.27 (s, 3H), 1.30 (t, 3H), 3.12 (m, 1H), 3.28 (m, 1H), 3.66 (s, 3H), 3.78 (m, 2 H), 5.20 (s, 1 H), 5.29 (d, 1 H).

(7mmols (7-methylhydrogen 9,11α-epoxy-3-ethoxy-17-hydroxy-17α-pregan-4-ene-7α, 21-dicarboxylate, γ-lactone) prepared in the previous step (18 mmols) 8 g portions of) were dissolved in 120 mL of 1,4-dioxane. The solution was combined with a mixture of 6.8 g of 53% m-chloroperbenzoic acid (20.9 mmols), 18.5 mL of 1.0 N sodium hydroxide (18.5 mmols) and 46 mL of dioxane / water (9: 1). The temperature was maintained at -3 ° C and the mixture was stirred for 2 hours. The temperature was raised to 25 ° C. and stirring continued for another 20 hours. The mixture was extracted four times with 100 mL portions of methylene chloride each time. A portion of the combined methylene chloride was dried over magnesium sulfate and the supernatant solvent was removed under vacuum distillation. The remaining 13.9 g was triturated with 50 mL ethyl ether to give 2.0 g white solid. A portion of 2.4g of the solid was chromatographed with 100g Merck silica gel (60μ). After initial washing with 1 L of 1: 1 ethyl acetate / heptane, the product was extracted with 7: 3 ratio of ethyl acetate / heptane. The enriched fractions were combined on a TLC generation basis (EMF plate; ethyl acetate / heptane 7: 3 (v / v) extractant; SWUV confirmed). In addition, 0.85 g of abundant material was obtained and recrystallized from 10 mL of isopropanol to obtain 0.7 g of 7-methylhydrogen 6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregan-4-ene-7α. , 21-dicarboxylate and γ-lactone were obtained. More contaminated fractions combined were 0.87 g of raw 7-methylhydrogen 6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid. and γ-lactone were obtained. This material was chromatographed on 67.8 g of Merck silica gel (40-63 μ). An additional 0.69 g of product was recovered with toluene containing 0.5 to 2.5% methanol.

7-Methylhydrogen 6β, 17-dihydroxy-9,11α-epoxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone:

Theoretically: C, 66.96 and H, 7.02: C, 66.68 and H, 7.16

¹ H NMR (CDCl ₃ ) ppm 1.06 (s, 3H), 1.36 (dm, 1H), 1.63 (s, 3H), 2.92 (m, 1H), 3.02 (dd, 1H), 3.12 (d, 1H), 3.64 (s, 3H), 4.61 (d, 1H), 5.96 (s, 1H).

¹³ C NMR (CDCl ₃ ) ppm 16.17, 21.32, 21.79, 24.36, 27.99, 28.94, 30.86, 31.09, 32.75, 33.19, 34.92, 36.77, 39.16, 43.98, 47.74, 51.56, 51.66, 65.36, 72.23, 94.79, 165.10, 171.36, 176.41, 199.59.

Example 47I

7-Methylhydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregan-4-ene-7β, 21-dicarboxylate, γ-lactone Manufacturing.

2 g (4.8 mmol) of 7-methylhydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate prepared by the method of Example 43 To γ-lactone was added 3.3 mL (14.4 mmol) of 25% sodium methoxide in methanol. The resulting yellow suspension was heated to 50 ° C. The solid did not dissolve. To the mixture was added 3.3 ml of methanol (Aldrich anhydride). The mixture was heated to reflux (65 ° C.) and homogenized. After 30 minutes the solid precipitate impeded stirring.

About 25 ml of anhydrous methanol was added and the mixture was transferred to a 100 mL flask. The mixture was heated at reflux for 16 hours where the mixture was dark and homogenous. The mixture was cooled to 25 ° C. and 70 ml of 3N HCl was added (exotherm). Several grams of ice were added to cool the mixture and the solution was extracted with two successive 25 mL portions of methylene chloride. The dark solution was dried over sodium sulfate and 2.5 cm pad of silica gel (E.Merck, 70-230 mesh 60 Filtered through the pore size). Silica was extracted with 100 mL of methylene chloride. The extracted methylene chloride was then concentrated in vacuo to give 1 g of brown foam crystallized upon addition of ethyl acetate. The silica pad was extracted second with 100 ml of 10% ethyl acetate / methylene chloride and the extracted solution was concentrated to give 650 mg of brown foam.

Thin layer chromatography (E.Merck, 60 F-254 silica gel 0.25 mm, toluene / ethyl acetate (1: 1, v / v)) showed 7-methylhydrogen 9,11α-epoxy-17-hydroxy in both samples. -3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone and 7-methylhydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregan The presence of -4-ene-7β, 21-dicarboxylate, γ-lactone was revealed but very few 7α-carboxypimers were present in the first sample. The first sample was softened with hot ethyl acetate (77 ° C.) and allowed to cool to 25 ° C. The mixture was filtered to give mp 254-258 ° C., 400 mg of a off white solid. H, ¹³ C and ¹³ C-APT matched the designated structure. A small amount of ethyl acetate remained in the sample but HPLC (Zorbax SB-C8 150 × 4.6nn, 2 mL / min, isocratic 40:60 (v / v) A: B, A = acetonitrile / methanol (1: 1) , B = water / 0.1% trifluoroacetic acid, detection at 210 nm) (HPLC showed 98.6 region percent), and TLC (toluene-ethylacetate 1: 1, v / v) showed no raw material.

FAB-MS confirmed the molecular weight of 414 with M.H at 415.2.

¹ H NMR (400 MHz, Deuterochloroform) σ 0.95 (s, 3H), 1.50 (s, 3H), 1.45 (m, 3H), 1.55-2.7 (m, 15H), 2.85 (t, J = 13, 1H ), 3.25 (d, J = 6, 1 H), 3.65 (s, 3 H), 5.78 (s, 1 H).

Example 47J

9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylic acid, γ-lactone Manufacturing.

774 mg (1.82 mmol) of 7-methylhydrogen 9,11α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene- prepared by the method of Example 43 and suspended in 3 ml of acetonitrile. 3 mL (7.5 mmol, 2.0 equiv) of 2.5 M sodium hydroxide was added to 7α, 21-dicarboxylate and γ-lactone. The mixture turned yellow and homogenized after 10 minutes.

To monitor the progress of the reaction, an aliquot (0.1 mL) of the mixture was quenched in 0.01 mL of 3M sulfuric acid and extracted in 4 mL glass vials with ethyl acetate (0.2 mL). The phases were separated by pipetting off the lower aqueous phase. The organic phase was stripped and the residue analyzed by HPLC using the method described in Example 47H. There was little change in composition of the mixture at 25 ° C. after 50 minutes.

The mixture was heated to reflux (about 90 ° C.) for 50 minutes. HPLC analysis of the mixture showed 6 zone percent of remaining raw material. The mixture was stirred at 25 ° C. for 65 h. Acidification, extraction and HPLC analysis of the aliquots as described above confirmed that no raw material remained.

The mixture was strongly acidified with about 4 mL of 3M sulfuric acid and extracted with two portions of methylene chloride (about 10 mL). The organic phases were combined and dried over sodium sulfate. Concentration on a rotary evaporator resulted in 780 g of solid. The solid was recrystallized from dimethylformamide / methanol to give 503 mg (67%) of a tan crystalline solid. The sample melted near 260 ° C. with a gas when heated rapidly. When the sample was slowly heated to 285 ° C., the sample slowly became dark and became a solid.

¹ H NMR (dimethylsulfoside d-6, 400 MHz) σ 0.85 (s, 3H), 1.4 (s, 3H), 1.3-2.9 (m, 19H), 3.15 (m, 1H), 5.55 (s, 1H) , 11.8 (br, 1 H).

Example 47K

Scheme 1: Step 3D: Method I: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

2 equivalents of dipotassium phosphate, 3 equivalents of chlorodifluoroacetamide and 22 equivalents of hydrogen peroxide (30 equivalents) of a 0.2 M ester solution of formula IIA in methylene chloride dissolved in the same amount of water (50% w / w aqueous solution) % Aqueous solution). The mixture was stirred at 25 ° C. for 23 h. The reaction was diluted with the same amount of water as the hydrogen peroxide charge and methylene chloride was separated. A portion of methylene chloride was washed once with 3% sodium sulfite solution (volume corresponding to 1.75 times the hydrogen peroxide charge). A portion of methylene chloride was separated and dried over sodium sulfate. The solution was concentrated under atmospheric distillation until a head temperature of 70 ° C. was achieved. The residue was purified by HPLC, ₁ H and ¹³ C. Evaluated via NMR (CDCl ₃ ). The yield of epoxymexrenone was determined to be 54.2 area% via HPLC.

Example 47L

Scheme 1: Step 3D: Method J: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

The procedure of Example 47K was repeated using heptafluoroacetamide (CF ₃ CF ₂ CF ₂ CONH ₂ ) instead of chlorodifluoroacetamide. The yield of epoxymexrenone was determined to be 58.4 area% via HPLC.

Example 48 Epoxidation of Esters of Formula (IIA) Using Toluene

Scheme 1: Step 3D: Method J: Synthesis of methylhydrogen 9,11α-epoxy-17α-hydroxy-3-oxopregan-4-ene-7α, 21-dicarboxylate, γ-lactone.

The ester of formula (IAA) was converted to epoxymexrenone by the method described generally in Example 46 except that toluene was used as the solvent. Materials charged to the reactor include ester (2.7 g) trichloroacetamide (2.5 g), dipotassium hydrogen phosphate (1.7 g), hydrogen peroxide (17.0 g) and toluene (50 ml). The reaction was allowed to exothermic to 28 ° C. and was completed after 4 hours. The three phase mixtures obtained were cooled to 15 ° C., filtered, washed with water and dried to give 2.5 g of product.

Example 49-Scheme 4: Method A: Epoxidation of 9,11-Dienone

A compound named XVIIA (a compound having both -AA- and -BB-, -CH ₂ -CH _2- ) (40.67 g) was dissolved in methylene chloride (250 mL) in a 1 liter three-necked flask and externally iced with a mixture of ice salts. Cooled. After 30% hydrogen peroxide (200 g) was slowly added over 1 hour, dipotassium phosphate (22.5 g) and trichloroacetonitrile (83.5 g) were added thereto, and the mixture was cooled to 2 ° C. The reaction mixture was stirred at 12 ° C. for 8 hours and at room temperature for 14 hours. A small amount of the organic layer was recovered and confirmed as a result of any raw material enon <0.5%. Water (400 mL) was added, stirred for 15 minutes and the layers separated. The organic layer was washed successively with 200 mL of potassium iodide (10%), 200 mL of sodium thiosulfate (10%) and 100 mL of saturated sodium bicarbonate solution each time separating the layers. The organic layer was dried over anhydrous magnesium sulfate and concentrated to give an original epoxide (41 g). Crystallization from ethyl acetate: methylene chloride afforded 14.9 g of pure material.

Example 50-Scheme 4: Method B: Epoxidation of Compound WVIIA with m-chloroperbenzoic acid

Compound WVIIA (18.0 g) was dissolved in 250 mL of methylene chloride and cooled to 10 ° C. Under stirring solid m-chloroperbenzoic acid (50-60% pure, 21.86 g) was added over 15 minutes. No rise in temperature was observed. The reaction mixture was stirred for 3 hours and checked for the presence of dienone. The reaction mixture was treated successively with sodium sulfite solution (10%), sodium hydroxide solution (0.5N), hydrochloric acid solution (5%) and finally 50 mL of saturated brine solution. After drying and evaporating with anhydrous magnesium sulfate, 17.64 g of epoxide was obtained and used directly in the next step. The product was found to contain Baeyer-Villiger oxidation products which must be removed by softening from ethyl acetate followed by crystallization from methylene chloride. To a 500 g size, the precipitated m-chlorobenzoic acid was filtered off and then subjected to normal finishing.

Example 51-Scheme 4: Method C: Epoxidation of compound WVIIA with trichloroacetamide.

Compound WVIIA (2 g) was dissolved in 25 mL of methylene chloride. Trichloroacetamide (2 g) and dipotassium phosphate (2 g) were added. Under stirring, 30% hydrogen peroxide (10 mL) was added at room temperature and stirring was continued for 18 hours to give epoxide (1.63 g). No Baeyer-Villiger product was formed.

Example 52

Potassium hydroxide (56.39 g; 1005.3 mmol; 3.00 eq.) Was charged to a 2000 mL flask and suspended with dimethyl sulfoxide (750.0 mL) at ambient temperature. Flask with THF (956.0 mL) trienone corresponding to Formula XX (R ³ is H and -AA- and -BB- are -CH ₂ CH _2- ) (100.00 g; 335.01 mmol; 1.00 eq) Filled in. Trimethylsulfonium methyl sulfate (126.14 g; 670.02 mmol; 2.00 eq.) Was placed in a flask and the resulting mixture was heated at reflux, i.e. at 80-85 ° C. for 1 hour. Conversion to 17-spirooxymethylene was confirmed by HPLC. Water (460 mL) was charged over 30 minutes and then about THF 1 L was stripped from the reaction mixture under vacuum while cooling the reaction mixture to 15 ° C. The resulting mixture was filtered and the solid oxirane product was washed twice with 200 mL aliquots of water. The product was observed to crystallize very well and filtration was carried out. The product was then dried at 40 ° C. under vacuum. 104.6 g of 3-methylenolether Δ-5,6,9,11, -17-oxirane steroid product was isolated.

Example 53

Sodium ethoxide (41.94 g; 616.25 mmol; 1.90 eq.) Was charged to a dry 500 mL reactor under a blanket of nitrogen. Ethanol (270.9 mL) was charged to the reactor and sodium methoxide was slurried in ethanol. Diethylmalonate (103.90 g; 648.68 mmol; 2.00 eq.) Was added to the slurry after addition of the oxiranesteroid prepared by the method described in Example 52 (104.60 g; 324.34 mmol; 1.00 eq.) And the resulting mixture was refluxed. That is, it heated to 80-85 degreeC. The completion of the reaction was confirmed by HPLC and heating continued for 4 hours. Water (337.86 mL) was charged to the reaction mixture over 30 minutes while the mixture was cooled to 15 ° C. After stirring was continued for 30 minutes, the reaction slurry was filtered while preparing a filter cake composed of fine amorphous powder. The filter cake was washed twice with water (200 mL) and then dried in vacuo to ambient temperature. 133.8 g of 3-methylenolether-Δ5,6,9,11, -17-spirolactone-21-ethoxycarbonyl intermediate were isolated.

Example 54

3-Methylenolether-Δ5,6,9,11, -17-spirolactone-21-ethoxycarbonyl intermediate (R ³ is H and -AA- and -BB- are as prepared in Example 53, respectively. -CH ₂ -CH ₂ -Formula WVIII; 133.80 g; 313.68 mmol; 1.00 eq) was charged to the reactor with sodium chloride (27.50 g; 470.52 mmol; 1.50 eq.) And dimethylformamide (709 mL) and water (5 mL) were added. Into a 2000 mL reactor under stirring. The resulting mixture was heated to reflux, ie, 138-142 ° C. for 3 hours after completion of the reaction by HPLC. Water was then added to the mixture over 30 minutes while the mixture was cooling to 15 ° C. After the reaction slurry was filtered, stirring was continued for 30 minutes to recover an amorphous solid reaction product which is a filter cake. The filter cake was dried and washed twice (200 mL aliquots of water). The product 3-methylenolether-17-spirolactone was dried to give 91.6 g (82.3% yield; 96 area% quantification).

Example 55

Enolether, ethanol (250 mL), acetic acid (250 mL) and water (250 mL) prepared as in Example 54 (91.60 g; 258.36 mmol; 1.00 eq.) Were charged to a 2000 mL reactor and the resulting slurry was heated to reflux for 2 hours. . Water (600 mL) was charged over 30 minutes while the reaction mixture was cooled to 15 ° C. The reaction slurry was then filtered and the filter cake was washed twice with water (200 mL aliquots). The filter cake was dried; 84.4 g of the product 3-keto Δ4,5, 9,11, -17-spirolactone were isolated (R ³ is H and -AA- and -BB- are -CH ₂ -CH _2- ; 95.9% yield).

Example 56

Compound XVIIA (1 kg; 2.81 moles) was charged to a 22 L four-necked flask with carbon tetrachloride (3.2 L). N-bromo-succinamid (538 g) followed by acetonitrile (3.2 L) was added to the mixture. The resulting mixture was heated to reflux and maintained at 68 [deg.] C. reflux for about 3 hours to give a clear orange solution. After 5 hours of heating, the solution turned dark. After 6 hours the heat was removed and the reaction mixture was sampled. The solvent was stripped under vacuum and ethyl acetate (6 L) was added to the residue at the bottom of the distiller. The resulting mixture was stirred after adding 5% sodium bicarbonate solution (4 L), allowing the phases to separate, and stirred for 15 minutes. The aqueous layer was removed and saturated brine solution (4 L) was injected into the stirred mixture for 15 minutes. The phases were separated again and the organic layer was stripped under vacuum to produce a thick slurry. Dimethylformamide (4 L) was then added and stripping was continued to a pot temperature of 55 ° C. The bottom of the distiller was left overnight and DABCO (330 g) and lithium bromide (243 g) were added. The mixture was then heated to 70 ° C. An hour and a half after heating, additional DABCO (40 g) was added to the liquid chromatography sample after 3.50 hours of heating. After 4.5 hours of heating, water (4 L) was injected and the resulting mixture was cooled to 15 ° C. The slurry was filtered and the cake washed with water (3 L) and dried in the filter overnight. The wet cake (978 g) was charged back into the 22 L flask and dimethylformamide (7 L) was added. The resulting mixture was also heated to 105 ° C. at the point where the cake was completely filled into solution. The heat source was removed and the mixture in the flask was stirred and cooled. Ice water was applied to the reactor jacket to cool the mixture in the reactor to 14 ° C. and left for 2 hours. The resulting slurry was filtered and washed twice with a 2.5 L aliquot of water. The filter cake was dried under vacuum overnight. 510 g of a light brown solid product were obtained.

Example 57

The 2-L four-necked flask was charged with the following: 9,11-epoxycanenone (100.00 g; 282.1 mmol; 1.00 eq), dimethylformamide (650.0 mL), lithium chloride (30.00 g; 707.0 mmol; 2.51 eq.) And acetonecyanohydrin (72.04 g; 77.3 mL; 846.4 mmol; 3.00 eq.). The resulting suspension was mechanically stirred and treated with tetramethylguanidine (45.49 g; 49.6 mL; 395.0 mmol; 1.40 eq.). The system was then filtered through a water cooled and dry ice tumbler (filled with acetone with dry ice) to prevent the escape of HCN. Ventane was passed from a dry ice tumbler into a scrubber filled with a large amount of excess chlorine bleach. The mixture was heated to 80 ° C.

After 18 hours, stirring gave a dark red-brown solution that was cooled to room temperature. During the cooling process, nitrogen was sparged into the solution to remove the remaining HCN along with the vent line passed into the bleach in the scrubber. After two hours the solution was treated with acetic acid (72 g) and stirred for 30 minutes. The original mixture was poured into ice water (2 L) with stirring. The stirred suspension was further treated with 10% aqueous HCl and stirred for 1 hour. The mixture was then filtered to give a red solid (73 g), like dark brick. The filtrate was placed in a 4 L separatory funnel and extracted with methylene chloride (3 x 800 mL); The organic layers were combined and back extracted with water (2 × 2 L). The methylene chloride solution was concentrated in vacuo to yield 61 g of dark red oil.

After leaving the aqueous wash fractions overnight, a significant precipitate occurred. This precipitate was recovered by filtration and identified as pure product enamine (14.8 g).

After drying, the original red solid (73 g) was analyzed by HPLC and determined that the main component was 9,11-epoxyamine. HPLC also indicated that enamine is the main component of the red oil obtained from the methylene chloride finish. The calculated molarity of enamine is 46%.

Example 58

Prepared 9,11-epoxyamine (4.600 g; 0.011261 mol; 1.00 eq.) As in Example 57 was injected into a 1000 mL round bottom flask. Methanol (300 mL) and 0.5 wt% aqueous HCl (192 mL) were added to the heated mixture at reflux for 17 h. Methanol was then removed under vacuum to subtract the amount of material in the still pot to 50 mL, resulting in the formation of a white precipitate. Water (100 mL) was then added to the filtered slurry to produce a white solid cake washed three times with water. The yield of solid 9,11-epoxydiketone product was 3.747 g (81.3%).

Example 59

Epoxydiketone (200 mg; 0.49 mmol) prepared as in Example 58 was suspended in methanol (3 mL) and 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) was added to the mixture. It was. The mixture was homogenized upon heating at reflux for 24 hours. It was then concentrated to 30 ° C. on a rotary evaporator to dryness and the remainder partitioned between methylene chloride and 3.0 N HCl. Concentration of the organic phase resulted in a yellow solid (193 mg) determined with 22% by weight epoxymexrenone. Yield 20%.

Example 60

To 100 mg diketone (prepared as in Example 58) suspended in 1.5 mL methanol was added a 25% (w / w) sodium methoxide solution of 10 microliters (0.18 eq) in methanol. The solution was heated to reflux. After 30 minutes no diketone remained and 5-cyanoester was present. To the mixture was added a 46 microliter 25% (w / w) methanol sodium solution in methanol. The mixture was judged by HPLC and heated at reflux for 23 hours, the main product being epoxymexrenone.

Example 61

0.34 mL of triethylamine was added to 2 g of diketone (prepared as in Example 48) suspended in 30 ml of dry methanol. The suspension was heated at reflux for 4.5 h. The mixture was stirred at 25 ° C. for 16 h. The obtained suspension was filtered to obtain 1.3 g of 5-cyanoester as a white solid.

To 6.6 g of diketone suspended in 80 mL of methanol, 2.8 mL of triethylamine was added. The mixture was heated at reflux for 4 hours and stirred at 25 rpm for 88 hours when the product crystallized out of solution. Methanol wash followed by filtration gave 5.8 g of cyanoester as a white powder. The material was recrystallized from chloroform / methanol to give 3.1 g of crystalline material homogenized by HPLC.

In view of the above, it is seen that several objects of the present invention can be achieved and other beneficial results obtained.

As various changes are made in the composition and process without departing from the scope of the present invention, all of the problems shown in the illustrations contained in or accompanied by the above description will be construed as embodiments rather than in a limited sense.

New compounds

The present invention is further directed to additional polycyclic organic moieties useful as chromatography markers in the preparation of steroidal compounds with desirable bioactivity such as spironolactone or epoxymexrenone.

In summary, any compound consisting of a substituted or unsubstituted steroid nucleus and a substituted or unsubstituted carbocyclic ring conjugated to the 13,17 position of the steroid nucleus is internal to the preparation of steroidal materials such as spironolactone and epoxymexrenone. Or as a chromatographic marker. In particular, methyl compounds 2,3,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3aβ, 11aβ-dimethyl-1,9-dioxo-1H-pentaleno [ 1,6a-a] phenanthrene-6α-carboxylic acid:

Formula (C-1)

Is useful as a chromatography marker. One novel feature of these compounds and related compounds of the invention is the conjugated carbocyclic ring attached to the D ring of the steroid nucleus. Spironolactone and epoxymexrenone lack this property and instead have a 20-spirolactone ring.

As used herein, the steroid nucleus of a compound corresponds to the structure:

Formula (C-2)

This structure reflects conventional numbering and ring naming for steroid compounds. The steroid nucleus can be saturated, unsaturated, substituted or unsubstituted. Preferably, it comprises at least one to four unsaturated bonds. More preferably, the A, C and D rings each contain at least one unsaturated bond. The steroid nucleus may also be substituted as discussed in more detail below. Preferably, the nucleus is substituted with at least a C7 ester group.

As used herein, the carbocyclic ring conjugated to the steroid nucleus corresponds to four, five or six carbon cyclic backbones. It may be saturated, unsaturated, substituted or unsubstituted. Preferably, it is saturated or has one double bond and is substituted with a hydroxy or keto group. Moreover, the carbocyclic ring preferably has an α-orientation associated with the steroid nucleus.

In a preferred embodiment, the carbocyclic ring consists of five carbon cyclic backbones and has an α-orientation, and the compound is selected from the group consisting of compounds corresponding to the formula:

Formula C-3

Wherein -AA- represents a -CR ¹⁰² R ^102a -CR ¹⁰³ R ^103a -or -CR ¹⁰² = CR ¹⁰³ -group, where -CR ¹⁰² R ^102a -and -CR ¹⁰² = correspond to C2 carbon, and -CR ¹⁰³ R ^103a -and = CR ¹⁰³ -correspond to C3 carbon;

-DD- is -CHR ¹⁰⁴ -CH- or -CR ¹⁰⁴ = C- represents a group;

-EE- represents a -CH ₂ -CR ¹¹⁰ -or -CH = C- group;

-AE- represents a -CR ¹⁰² R ^102a -CH ₂ -or -CR ¹⁰² = CH group, where -CR ¹⁰² R ^102a -and -CR ¹⁰² = correspond to C2 carbon and -CH ₂ -and = CH- are C1 Corresponds to carbon;

-GG- represents a -CR ¹⁰⁶ R ^106a -CHR ¹⁰⁷ -or -CR ¹⁰⁶ = CR ¹⁰⁷ -group, where -CR ¹⁰⁶ R ^106a -and -CR ¹⁰⁶ = correspond to C6 carbon and -CHR ¹⁰⁷ -and = CR ¹⁰⁷ -Corresponds to C7 carbon.

-JJ- represents a -CR ¹⁰⁸ -CR ¹⁰⁹ -or -C = C- group, wherein -CR ¹⁰⁸ -corresponds to C8 carbon and -CR ¹⁰⁹ -corresponds to C9 carbon;

-LL- represents a -CR ¹¹¹ R ^111a -CH ₂ -or -CR ¹¹¹ = CH- group, where -CR ¹¹¹ R ^111a -and -CR ¹¹¹ = correspond to C11 carbon and -CHR ² -and = CH- Corresponds to C12 carbon;

-JL- is -CR ¹⁰⁹ -CR ¹¹¹ R ^111a - or -C = CR ¹¹¹ - represents a group, wherein -CR ¹⁰⁹ - and -C = correspond to the C9 carbon, and is, -CR ¹¹¹ R ^111a - and -CR ¹¹¹ - Corresponds to C11;

-MM- represents a -CR ¹¹⁴ -CH ₂ -or -C = CH- group, wherein -CR ¹¹⁴ -and -C = correspond to C14 carbon, and -CH ₂ -and -C = H- are for C15 carbon Corresponding;

-JM- represents a -CR ¹⁰⁸ -CR ¹¹⁴ -or -C = C- group, wherein -CR ¹⁰⁸ -corresponds to C8 carbon and -CR ¹¹⁴ -corresponds to C14 carbon;

-QQ- represents a group -CR ¹²⁰ R ^120a -CR ¹¹⁹ R ^119a -or -CR ¹²⁰ = CR ¹¹⁹ -where -CR ¹²⁰ R ^120a -and -CR ¹²⁰ = CR ¹¹⁹ -correspond to C20 carbon, and -CR ¹¹⁹ R ^119a -and = CR ¹¹⁹ -correspond to C19 carbon;

-QT- represents a group -CR ¹¹⁹ R ^119a -CHR ¹¹⁸ -or -CR ¹¹⁹ = CR ¹¹⁸ -where -CR ¹¹⁹ R ^119a -and -CR ¹¹⁹ = correspond to C19 carbon and -CHR ¹¹⁸ -and = CR ¹¹⁸ -Corresponds to C18;

R ¹⁰² is hydrogen, alkyl, alkenyl, or alkynyl;

R ^102a is hydrogen; Or -AA- represents a bond between a C2 carbon atom and a C3 carbon atom when -CR ¹⁰² = CR ¹⁰³ -group and -AE- represents -CR ¹⁰² R ^102a -CH ₂ -group; Or -AE- represents a bond between a C1 carbon atom and a C2 carbon atom when -AA- represents -CR ¹⁰² = CH- group and -AA- represents -CR ¹⁰² R ^102a -CR ¹⁰³ R ^103a -group;

R ¹⁰³ is hydrogen, hydroxy, protected hydroxy, R ¹⁰³ O-, R ¹⁰³ C (O) O-, Forms oxo with R ¹⁰³ OC (O) 0-, or R ^103a ; However, when R ¹⁰³ together with R ^103a form an oxo -AA- is ^{-CR 102 R 102a -CR 103 R 103a} - is;

R ^103a forms oxo with hydrogen or R ¹⁰³ ; With the proviso that when R ^103a together with R a to form an oxo -AA- ¹⁰³ is ^{-CR 102 R 102a -CR 103 R 103a} - is;

R ¹⁰⁴ is hydrogen, alkyl, alkenyl or alkynyl;

R ¹⁰⁶ forms oxo with hydrogen, hydroxy or protected hydroxy, or R ^106a , or together with R ^106a and the attached carbon atom form a cyclopropyl, cyclobutyl or cyclopentyl ring; However, when R ¹⁰⁶ form an oxo with R ^106a -GG- is ^{-CR 106 R 106a -CR 107 R 107a} - a.

R ^106a forms oxo with hydrogen, hydroxy or protected hydroxy, or R ^106a , or forms a cyclopropyl, cyclobutyl, or cyclopentyl ring with R ¹⁰⁶ and the attached carbon atom, or R ¹⁰⁷ and R ^106a with attached carbon atom forms a cyclopropyl, cyclobutyl, or cyclopentyl ring;

R ¹⁰⁷ is hydrogen; hydroxycarbonyl; Lower alkyl, alkenyl, alkynyl, aryl, heteroaryl or aralkyl; Haloalkyl; Hydroxyalkyl; Alkoxyalkyl; Lower alkanoyl, alkenoyl, alkinoyl, aryl oil, heteroaryl oil or aralkanoyl; Lower alkoxycarbonyl, alkenoxycarbonyl, alkynoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl or aralkyloxycarbonyl; Lower alkanoylthio, alkenoylthio, alkinoylthio, aryloilthio, heteroaroylthio or aralkanoylthio; Lower alkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio or aralkylthio; Carbamyl; Alkoxycarbonylamino; Or cyano; or

R ¹⁰⁷ with carbon attached to R ^106a form a cyclopropyl, cyclobutyl or cyclopentyl ring; or

R ¹⁰⁷ and R ¹¹⁴ together with C7, C8 and C14 carbon atoms form γ-lactone;

R ¹⁰⁸ is hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl, R ¹⁴⁰ O-, R ¹⁴⁰ C (O) 0-, or R ¹⁴⁰ 0C (O) 0-; Or -JJ- represents a bond between a C8 carbon atom and a C9 carbon atom when -JM- represents a -C = C- group and -JM- represents a -CR ¹⁰⁸ -CR ¹¹⁴ -group; Or a bond between a C8 and C14 carbon atom when -JM- represents a -C = C- group and -JJ- represents a -CR ¹⁰⁸ -CR ¹¹⁴ -group.

R ¹⁰⁹ is hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl, R ¹⁵⁰ O—, R ¹⁵⁰ C (O) O—, or R ¹⁵⁰ OC (O) O—; Or -JL- represents a bond between a C9 carbon atom and a C11 carbon atom when -C = CR ¹¹¹ -group and -JJ- represent a -CR ¹⁰⁸ -CR ¹⁰⁹ -group; Or -JJ- represents a bond between a C9 carbon atom and a C8 carbon atom when the -C = C- group -JL-group represents a -CR ¹⁰⁹ -CR ¹¹¹ R ^111a -group.

R ¹¹⁰ is hydrogen or methyl;

R ¹¹¹ is hydrogen, hydroxy or protected with a hydroxy, or R ^111a form an oxo, it provided that, when R ^111a form an oxo with -JL- is -CR ¹⁰⁹ -CR ¹¹¹ R ^111a - group, - LL- represents a -CR ¹¹¹ R ^111a -CH ₂ -group;

R ^111a is hydrogen, or forms an oxo together with R ^111, however, when the R ^111a form an oxo with R ¹¹¹ -JL- is -CR ¹⁰⁹ -CR ¹¹¹ R ^111a - group, a -CR -LL- ¹¹¹ R ^111a -CH ₂ -group; Or -JL- represents a bond between a C11 carbon atom and a C9 carbon atom when -C = CR ¹¹¹ -group and -LL- represent -CR ¹¹¹ R ^111a -CH ₂ -group; Or R ^111a is -LL- the group -CR ¹¹¹ = CH-, -JL- is ^{-CR 109 R 109a -CR 111 R 111a} - represents a bond between the C11 carbon atom and the C12 to represent a carbon atom;

R ¹¹⁴ is hydrogen, hydroxy, protected hydroxy, alkyl, alkenyl, alkynyl, R ¹⁶⁰ O-, R ¹⁶⁰ C (0) 0-, R ¹⁶⁰ OC (0) 0-; Or R ¹¹⁴ and R ¹⁰⁷ together with C7, C8 and C14 carbons form γ-lactone; Or R ¹¹⁴ represents a bond between a C14 carbon atom and a C8 carbon atom when -JM- represents -C = C- group and -MM- represents -CR ¹¹⁴ -CH ₂ -group; Or R ¹¹⁴ represents a bond between a C14 carbon atom and a C15 carbon atom when -MM- represents -C = CH- group and -JM- represents -CR ¹⁰⁸ -CR ¹¹⁴ -group;

R ¹¹⁸ is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylthio, alkenylthio or cyano;

R ^118a is hydrogen or represents a bond between a C18 carbon atom and a C19 carbon atom when -QT- represents -CR ¹¹⁸ = CR ¹¹⁹ -and -QQ- represents -CR ¹¹⁹ R ^119a -CR ¹²⁰ R ^120a- ;

R ¹¹⁹ is hydrogen, alkyl or alkenyl;

R ^119a is hydrogen or represents a bond between a C19 carbon atom and a C20 carbon atom when -QQ- represents -CR ¹²⁰ = CR ¹¹⁹ -group and -QT- represents -CR ¹¹⁹ R ^119a -CR ¹¹⁸ R ^118a -group ; Or -QT- represents a bond between a C19 carbon atom and a C18 carbon atom when -CR ¹¹⁹ = CR ¹¹⁸ -group and -QQ- represents -CR ¹¹⁹ R ^119a -CR ¹²⁰ R ^120a -group;

R ¹²⁰ forms oxo with hydrogen, hydroxy, protected hydroxy or R ^120a ; Provided that when R ¹²⁰ forms oxo with R ^120a , -QQ- represents a -CR ¹¹⁹ R ^119a -CR ¹²⁰ R ^120a -group;

R ^120a forms oxo with hydrogen or R ¹²⁰ ; Provided that when R ^120a forms oxo with R ¹²⁰ , -QQ- represents a -CR ¹¹⁹ R ^119a -CR ¹²⁰ R ^120a -group; And

R ¹³⁰ , R ¹⁴⁰ , R ¹⁵⁰ and R ¹⁶⁰ is alkyl, alkenyl, alkynyl, aryl or heteroaryl, respectively.

More preferably, the compound is R ¹⁰⁷ is hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoylthio, lower alkylthio, carbamyl or with R ^106a and attached carbon R ¹⁰⁷ forms a cyclopropyl ring; R ¹⁰⁶ is hydrogen, hydroxy or together with R ^106a and the attached carbon form a cyclopropyl ring; R ^106a is hydrogen, hydroxy or together with R ¹⁰⁶ and the attached carbon form a cyclopropyl ring; Or R ^106a together with R ¹⁰⁷ and the attached carbon form a cyclopropyl ring; R ¹²⁰ corresponds to a compound of formula C-3 which is keto.

The following definitions apply to the discussion relating to the 13,17-conjugated ring compounds disclosed in the instant embodiments.

The term "lower alkyl" refers to alkyl radicals having from 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl. The radical may be straight, branched chain, or cyclic and substituted (particularly with aryl), unsubstituted or heterosubstituted.

The term "lower alkanoyl" means a radical derived from straight-chain alkyl having preferably 1 to 7 carbon atoms and attached to the parent molecular moiety via a carbonyl group.

The term "lower alkoxycarbonyl" means a radical derived from straight-chain alkyl having preferably 1 to 7 carbon atoms and attached to an oxygen atom, which oxygen atom is attached to the parent molecular moiety via a carbonyl group . Especially preferred are methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl and n-hexyloxycarbonyl.

The term "lower alkenyl" refers to alkenyl having 2 to 6 carbon atoms such as ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, sec-butenyl and tert-butenyl, pentenyl and hexenyl Means radicals. The radical is a straight or branched chain and may be substituted, unsubstituted or heterosubstituted. The terms "lower alkenoyl" and "lower alkenoxycarbonyl" are defined in the same way as "lower alkanoyl" and "lower alkoxycarbonyl", except that they are derived from straight chain alkenyl instead of straight chain alkyl. do. Preferably, the oxygen attached to the alkenyl radical of any alkenoxycarbonyl group is separated from the unsaturated carbon by at least one methylene group.

The term "lower alkynyl" refers to alkyes having 2 to 6 atoms such as ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, sec-butynyl and tert-butynyl, pentynyl and hexynyl Means a Neyl radical. The radical is a straight or branched chain and may be substituted, unsubstituted or heterosubstituted. The terms "lower alkinoyl" and "lower alkynoxycarbonyl" are used in the same way as "lower alkanoyl" and "lower alkoxycarbonyl" except that they are derived from straight chain alkynyl instead of straight chain alkyl. Is defined. Preferably, the oxygen attached to the alkynyl radical of any alkynoxycarbonyl group is also separated from any unsaturated carbon by at least one methylene group.

"Aryl" moieties preferably contain atoms from 5 to 15, alone or with several substituents, and include phenyl.

The term "lower alkylthio" means a radical derived from straight-chain alkyl having preferably 1 to 7 carbon atoms and attached to the parent molecular moiety via a sulfur atom. Particularly preferably methylthio.

The term "lower alkanothio" refers to radicals derived from straight-chained alkyl having preferably 1 to 7 carbon atoms and attached to the carbonyl group attached to the parent molecular moiety via a sulfur atom. Particularly preferably acetylthio.

The terms "lower alkenylthio" and "lower alkenoylthio" are defined in the same way as "lower alkylthio" and "lower alkanoylthio", except that they are derived from straight chain alkenyl instead of straight chain alkyl. . Preferably the sulfur atom of the alkenylthio group is distinguished from any unsaturated carbon by at least one methylene group.

The terms "lower alkylynthio" and "lower alkynylthio" are defined in the same manner as "lower alkylthio" and "lower alkanoylthio", except that they are derived from straight chain alkenyl instead of straight chain alkyl. do. Preferably the sulfur atom of the alkynylthio group is distinguished from any unsaturated carbon by at least one methylene group.

The term "carbamyl" refers to -NH ₂ -radicals attached to the parent molecular moiety through a carbonyl group. The carbamyl group may be mono-substituted or di-substituted and the substituents may include alkyl, alkenyl, alkynyl and aryl radicals.

 The groups defined above may be unsubstituted or further substituted. Such additional substituents include alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, carboxyalkyl, acyl, carboxy such as acyloxy, halo such as chloro or fluoro, haloalkoxy, nitro, amino, amido and keto It may include. In addition to the additional substituents, the groups defined above may also include oxygen, sulfur, phosphorus and / or nitrogen.

"Me" as used herein means methyl; "Et" means ethyl; "Ac" means acetyl.

In a still more preferred embodiment the compound is selected from the group consisting of compounds having the formula:

Formula (C-5)

Formula (C-6)

Formula (C-7)

Formula (C-8)

Formula (C-9)

Formula (C-10)

Formula (C-11)

Formula (C-12)

Formula (C-13a)

Formula C-13b

Formula (C-13c)

Formula C-13d

And

Formula (C-13e)

Wherein R ¹⁰⁷ , R ¹⁰⁶ , R ^106a and R ¹²⁰ are as defined above. Preferably, R ¹⁰⁷ is hydrogen, hydroxycarbonyl, lower alkyl, lower alkanoyl, lower alkoxycarbonyl, lower alkanoylthio, lower alkylthio, carbamyl or a cyclopropyl ring with R ^106a and the attached carbon atom. To form; R ¹⁰⁶ is hydrogen, hydroxy or together with R ^106a and the attached carbon atom form a cyclopropyl ring; R ^106a is hydrogen, hydroxy or together with R ¹⁰⁶ and the attached carbon atom form a cyclopropyl ring; Or together with R ¹⁰⁷ and the attached carbon atom form a cyclopropyl ring; And R ¹²⁰ is keto. Even more preferably, the compound further corresponds to the compound of formula C-5.

In an even more preferred embodiment, the compound of formula C-3 is selected from the group consisting of:

Formula C-14 Formula C-15

Ethyl 3 ', 4', 5 ', 17-propyl 3', 4 ', 5', 17-tetrahydro-

Tetrahydro-17β-methyl-17β-methyl-3,5'-

53,5'-dioxocyclopenta [13R, 17] -18-

Dioxocyclopenta [13R, 17] -norandrostar-4,8,14-triene-7α-

18-Norandrostar-4,8,14-carboxylic acid

Triene-7α-carboxylic acid

Formula C-16 Formula C-17

Butyl 3 ', 4', 5 ', 17- 1-methylethyl 3', 4 ', 5', 17-

Tetrahydro-17β-methyl-tetrahydro-17β-methyl-3,5'-

3,5'-dioxocyclopenta [13R, 17] -18-

Dioxocyclopenta [13R, 17] -norandrostar-4,8,14-triene-7α-

18-Norandrostar-4,8,14-carboxylic acid

Triene-7α-carboxylic acid

Formula C-18 Formula C-19

2-methylpropyl 1-methylpropyl 3 ', 4', 5 ', 17-

3 ', 4', 5 ', 17-tetrahydro-tetrahydro-17β-methyl-3,5'-

17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-

Dioxocyclopenta [13R, 17] -norandrostar-4,8,14-triene-7α-

18-Norandrostar-4,8,14-carboxylic acid

Triene-7α-carboxylic acid

Formula C-20 Formula C-21

Cyclopentyl 3 ', 4', 5 ', 17-hexyl 3', 4 ', 5', 17-tetrahydro-

Tetrahydro-17β-methyl-17β-methyl-3,5'-

3,5'-dioxocyclopenta [13R, 17] -18-

Dioxocyclopenta [13R, 17] -norandrostar-4,8,14-triene-7α-

18-Norandrostar-carboxylic acid

1,4,8,14-tetraene-7α-

Carboxylic acid

Formula C-22 Formula C-23

       Methyl 3 ', 4', 4 ', 17-propyl 3', 4 ', 5', 17-tetrahydro-

       Tetrahydro-17β-methyl 17β-methyl-3,5'-

       3,5'-dioxocyclopenta [13R, 17] -dioxocyclopenta [13R, 17] -18-

       18-Norandrostar-1,4,8,14-Norandrostar-1,4,8,14-tetraene-

       Tetraene-7α-Carbolic Acid 7α-Carboxylic Acid

Formula C-24 Formula C-25

       Ethyl 3 ', 4', 5 ', 17- 1-methylethyl 3', 4 ', 5', 17-

       Tetrahydro-17β-methyl tetrahydro-17β-methyl-3,5'-

       3,5'-dioxocyclopenta [13R, 17] -dioxocyclopenta [13R, 17] -18

       18-Norandrostar-1,4,8,14-Norandrostar-1,4,8,14-tetraene-

       Tetraene-7α-carboxylic acid 7α-carboxylic acid

Formula C-26 Formula C-27

       Butyl 3 ', 4', 5 ', 17- 1-methylpropyl 3', 4 ', 5', 17-

       Tetrahydro-17β-methyl tetrahydro-17β-methyl-3,5'-

       3,5'-dioxocyclopenta [13R, 17] -18-

       Dioxocyclopenta [13R, 17] -norandrostar-1,4,8,14-tetraene-

       18-Nordrostar-1,4,8,14-7α-carboxylic acid

       Tetraene-7α-carboxylic acid

Formula C-28 Formula C-29

       2-Methylpropyl hexyl 3 ', 4', 5 ', 17-tetrahydro-

       3 ', 4', 5 ', 17-tetrahydro-17β-methyl 3,5'-dioxocyclopenta

       17β-methyl 3,5'-dioxocyclopenta [13R, 17] -18-norandrostar-1,4,8,

       [13R, 17] -18-Norandrostar-1,4,8, 14-tetraene-7α-carboxylic acid

       14-tetraene-7α-carboxylic acid

(Formula C-30) (Formula C-31)

       1,2,4bR (4bR *), 5,5aS *, 7, 4bR (4bR *), 5,5aS *, 7,7aR *, 8,9,11,

       7aR *, 8,9,11,12bS * -dodecahydro-12,12bS * -decahydro-7A, 12b-dime

       7a, 12b-dimethyl-10aR * -cyclo-propal [1] tyl-10R * -cyclopropal (1) pentaleno

       Pentaleno [1,6a-a] phenanthrene-3,10-dione [1,6a-a] phenanthrene-3,10-dione

Formula C-32 Formula C-33

       1,2,4bs (4bR *), 5,5aS *, 8,9,11,12,2 ', 3', 3'aα, 4 ', 6', 11'aα, 12 ',

       12bR * -dodecahydro-7a, 12b-dimethyl 13'-octahydro-3'aR, 3'a, 11'a-

       -10aS * -cyclopropal [1] pentaleno [1, dimethyl-13'aR * -spiro [cycloprop

       6a-a] phenanthrene-3,10-dione plate-1,7 '(9'H)-[1H] pentaleno [1,6, a

                                              -a] phenanthrene] -1 ', 9'-dione

Formula C-34 Formula C-35

       2 ', 3', 3'aα4 ', 6', 10 ', 11', 11'aα, 7α- (acetylthio) -3 ', 4'-dihydro-

       12 ', 13'-decahydro-3'aR, 3'a, 11'a 17-methyl-cyclopenta [13,17]-

       -Dimethyl-13'aR * -spiro [cyclopro 18-norandrostar-4,8,14-triene-

       Phan-1,7 '(9'H)-[1H] pentaleno [1,6a-a] 3,5' (2'H) -dione

       Pennantrene] -1 ', 9'-Dion

Formula C-36 Formula C-37

       3 ', 4'-dihydro-17-methyl-3', 4 ', 5,5', 6,17-hexahydro-17β

       7α- (methylthio) -cyclopenta [13, -methylcyclopenta [13R, 17] -18-nor

       17] -18-Norandro-4,8,14-Androstar-4,8,14-Trienne-3,5 '

       Trien-3,5 '(2'H) -dione (2'H) -dione

Formula C-38 Formula C-39

      3 ', 4', 5 ', 17-tetrahydro-6α-hydromethyl 3', 4 ', 5', 17-tetrahydro-

      Hydroxy-17β-methyl-3,5'-dioxocyclo 6α-hydroxy-17β-methyl-3,5'-di

      Penta [13R, 17] -18-norandrostar-4,8, oxocyclopenta [13R, 17] -18-nor

      14-triene-7α-carboxylic acid androstar-4,8,14-triene-7α-

                                              Carboxylic acid

Chemical Formula C-40 Chemical Formula C-41

       Ethyl 3 ', 4', 5 ', 17-tetrahydro-propyl 3', 4 ', 5', 17-tetrahydro

       6α-hydroxy-17β-methyl-3,5'-dioxo-6α-hydroxy-17β-methyl-3,5'-di

       Cyclopenta [13R, 17] -18-Norandrostar oxocyclopenta [13R, 17] -18-Nor

       4,8,14-triene-7α-carboxylic acid androstar-4,8,14-triene-7α-

                                              Carboxylic acid

Formula C-42 Formula C-43

       1-methylethyl butyl 3 ', 4', 5 ', 17-tetrahydro-

       3 ', 4', 5 ', 17-tetrahydro-6α-hydroxy-17β-methyl-3,5'-

       6α-hydroxy-17β-methyl-dioxocyclopenta [13R, 17] -18-

       3,5'-dioxocyclopenta [13R, norandrostar-4,8,14-triene-

       17] -18-Norandrostar-4,8,14 7α-carboxylic acid

       -Triene-7α-carboxylic acid

Formula C-44 Formula C-45

       1-methylpropyl 3 ', 4', 5 ', 17-tetra 3', 4 ', 5', 17-tetrahydro-17β-

       Hydro-6α-hydroxy-17β-methyl-3,5 'methylcyclopenta [13R, 17] -18-nor

       -Dioxocyclopenta [13R, 17] -18-noran androstar-4,8,14-triene-3,5 '

       Drostar-4,8,14-triene-7α-carboxylic acid (2'H) -dione

Formula C-46 Formula C-47

       (C-46) (C-47)

       3 ', 4', 5 ', 17-tetrahydro-6β-hydromethyl 3', 4 ', 5', 17-tetrahydro-

       Hydroxy-17β-methyl-3,5'-dioxocyclo 6β-hydroxy-17β-methyl-3,5'-

       Penta [13R, 17] -18-norandrostar-4,8, dioxocyclopenta [13R, 17] -18-

       14-triene-7α-carboxylic acid norandrostar-4,8,14-triene-

                                               7α-carboxylic acid

Formula C-48

       Ethyl 3 ', 4', 5 ', 17-tetrahydro-6β propyl 3', 4 ', 5', 17-tetrahydrate

       -Hydroxy-17β-methyl-3,5'-dioxocyclo-6β-hydroxy-17β-methyl-3,5 '

       Lofenta [13R, 17] -18-norandrostar-4,8, -dioxocyclopenta [13R, 17] -18-

       14-triene-7α-carboxylic acid norandrostar-4,8,14-triene-

                                               7α-carboxylic acid

Formula C-50 Formula C-51

       1-methylethyl cyclopentyl 3 ', 4', 5 ', 17-tetra

       3 ', 4', 5 ', 17-tetrahydro-6β-hydroxy hydro-17β-methyl-3,5'-dioxo

       -17β-methyl-3,5'-dioxocyclopenta [13R, cyclopenta [13R, 17] -18-nor

       17] -18-Norandro-4,8,14-triene Androstar-4,8,14-triene-7α-

       -7α-carboxylic acid carboxylic acid

Formula C-52 Formula C-1

       1-methylpropyl 3 ', 4', 5 ', 17-tetramethyl 2,2,3a, 4,6,7,9,10,11,11a,

       Hydro-6β-hydroxy-17β-methyl-3,5 '12,13-dodecahydro-3αβ, 11α

       -Dioxocyclopenta [13R, 17] -18-β-dimethyl-1,9-dioxo-1H-pentale

       Norandrostar-4,8,14-triene-7α- no [1,6a-a] phenanthrene-6α-carboxy

       Carboxylic acid

Formula (C-53)

       Butyl 3 ', 4', 5 ', 17-tetrahydro-6β

       -Hydroxy-17β-methyl-3,5'-dioxocyclo

       Penta [13R, 17] -18-Norandrostar-4,8,14-

       Triene-7α-carboxylic acid

Formula (C-201)

(7α, 13R, 17β) -3 ', 4', 5 ', 17-tetrahydro-14-hydroxy-17-methyl-3,5'-dioxo-γ-lactone, cyclopenta [13,17] -18-norandrostar-4,9 (11) -diene-7-carboxylic acid

Formula (C-202)

[13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-

Dimethylcyclopenta [13,17] gona-1,3,5 (10) -trien-5 '[2'H] -one

Formula (C-203)

[13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17

-Dimethylcyclopenta [13,17] gona-1,3,5, (10), 6-tetraen-5 '[2'H] -one

Formula C-204

[13S, 17β] -3 ', 4'-dihydro-17-methyl-cyclopenta [13,17] -18-

Yellow drostar-4,6,8 (14) -triene-3,5 '[2'H] -dione

In the most preferred embodiment the compound is methyl 2,2,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3αβ, 11αβ-dimethyl-1,9-dioxo-1H Pentaleno [1,6a-a] phenanthrene-6α-carboxylic acid:

Formula (C-1)

This compound of formula C-1 is a preferred chromatographic marker, particularly in the preparation of epoxymexrenones.

The novel compounds discussed herein may also exist in their salt form.

Preparation of New Compound

In general, the novel compounds just described can be obtained by reacting a steroid with the 20-spiroxane ring and steroid nucleus previously described above with trihalogenated alkanoic acid. Advantageously, the reagent further comprises an alkali metal salt of the alkanoic acid used. It is particularly preferred that the reagent consists of alkali metal salts thereof, such as trifluoroacetic acid and potassium trifluoroacetic acid. Moreover, desiccants such as trifluoroacetic anhydride are preferably used in the course of the reaction to reduce the free water present in the acid.

The steroid compound used as a raw material preferably has the following formula:

Formula C-3

(Wherein -AA-, -DD-, -EE-, -AE-, -GG-, -JJ-, -LL-, -JL-, -MM-, -JM-, -QQ- and -QT- Is as described above). Such starting materials can be prepared and / or isolated by methods similar to those described in the process for synthesizing epoxymexrenone of Scheme 1 as discussed above. Alternatively, the starting material may be a commercial product.

The initial concentration of the steroid compound of Formula C-3 is preferably at least about 0.1% by weight of the total reaction mixture, more preferably about 2% to about 20% by weight, and is about 5% to about 15% by weight. It is more preferable. It is preferred that an excess of trihaloalkanoic acid is present. When using trifluoroacetic anhydride, it should be present in a proportion of at least about 3% by weight of the total reaction mixture, more preferably from about 5% to about 25% by weight, most preferably from about 10% to about It should be present at a rate of 15% by weight.

In addition, the reaction temperature should be higher than room temperature (22 ° C.). Preferably, the reaction temperature is about 40 ° C. to 100 ° C., more preferably 50 ° C. to 80 ° C., still more preferably 60 ° C. to 70 ° C., and most preferably 60 ° C. to 65 ° C. Increasing the reaction temperature above about 70 ° C. increases the amount of byproduct C14 lactone produced in the reaction. The reaction time should preferably be at least about 30 minutes, more preferably from about 30 minutes to about 6 hours, even more preferably from about 45 minutes to about 4 hours, and most preferably from about 1 hour to 2 hours. In a preferred embodiment, the reaction time is about 1 hour to 2 hours and the reaction temperature is maintained at about 60 ° C.

Scheme S-1 below illustrates a particularly preferred embodiment of this method:

As described in the scheme below, condensed ring steroids having different substituents at various positions throughout the steroid can be prepared. Those skilled in the art know additional procedures and methods not specifically described herein for introducing various substituents at different positions of the steroid. The starting material may be a condensed ring steroid or a 20-spiroxane ring steroid. To simplify the description when the starting material is a condensed ring steroid, the following scheme employs a particular steroid or group of steroids as an exemplary starting material. However, it should be understood that by using different condensed ring steroids as starting materials, different condensed ring steroid derivatives or analogs can be produced in the same series of reactions. Similarly, to simplify the technique when the starting material is a 20-spiroxan ring steroid, a specific 20-spiroxan ring steroid is used as the starting material. However, it should be understood that by using different 20-spiroxane ring steroids as starting materials, other 20-spiroxane ring steroid derivatives or analogs can be produced in the same series of reactions.

Steroids with C7 carboxylic acid substituents can be prepared by saponification of steroids with C7 alkoxycarbonyl substituents, such as compounds of Formula C-1. The saponification reaction can be carried out by treating the starting steroid with a basic reagent such as sodium hydroxide or potassium hydroxide in a suitable solvent such as methanol, ethanol, isopropanol, etc. in the presence or absence of water at temperatures up to the boiling point of the solvent. Can be. As illustrated in Scheme S-2, saponifying a compound of Formula C-1 yields a carboxylic acid of Formula C-101.

Carboxylic acids such as C-101 can be used as starting materials to prepare steroids having C7 carboxylic ester substituents other than metanoate. Treatment of such carboxylic acids with alkylating agents such as halogenated alkyls in the presence of a base (such as sodium bicarbonate, sodium carbonate, potassium bicarbonate, or triethylamine) in a solvent such as dimethylformamide yields the desired ester. Examples of suitable alkylating agents include ethyl iodide, ethyl bromide, isopropyl iodide, hexyl iodide, benzyl bromide, allyl iodide and the like.

Carboxylic acid C-101 is also a suitable starting material for the synthesis of carbamyl. Treatment of carboxylic acid with chloroformate such as isobutyl chloroformate or ethyl chloroformate in the presence of a base yields mixed anhydrides. Treatment of the mixed anhydride with amines (such as dimethylamine, methylamine or benzylamine) yields carbamyl (R ₁ and R ₂ are substituents on the various amines).

Several modifications are made at the C7 position using unsaturated ketones such as compounds of formula C-105 (shown below in Scheme S-3) as starting material. Sulfides are synthesized by addition of a suitable thiol under basic conditions. Examples of suitable thiols include methyl mercaptan, ethyl mercaptan and the like. Suitable bases include piperidine, triethylamine and the like.

Treatment of unsaturated ketones (such as compounds of Formula C-105) with thioalkanoic acids such as thioacetic acid provides C7 thioacyl compounds such as acetylthio.

Treatment of unsaturated ketones (such as compounds of Formula C-105) with dimethylsulfonium methylide, produced by treating trimethylsulfonium halide with a suitable base (such as sodium hydride) in a suitable solvent, results in condensation C6, C7 cyclopropyl Substituents may be added.

These various synthetic schemes are illustrated in Scheme S-3 below:

Steroids with a C6 spirocyclopropyl ring are synthesized according to the procedure described in Scheme S-4 below. Enones, such as compounds of formula C-110, are first protected as C3 enol ethers by treatment with ortho esters such as triethyl ortho formate or trimethyl ortho formate in the presence of an acid such as p-toluenesulfonic acid. The addition of phosphorus oxychloride to dimethylformamide treats the resulting enol ether with the Vilsmeier reagent produced in situ to provide a formyl compound such as the compound of formula C-112. Reduction of the formyl group is carried out in a solvent such as tetrahydrofuran using a hydride reducing agent such as lithium tri-tert-butoxyaluminum hydride. This produces an intermediate alcohol, which is treated with an acid and the water is removed to give a 6-methylene compound, such as the compound of formula C-113. Suitable acids include hydrochloric acid in an aqueous medium. Treatment of the 6-methylene compound with diazomethane yields the intermediate pyrazoline, which is decomposed by heating to give a spirocyclopropyl compound, such as the compound of formula C-114. Protected enol ethers (such as compounds of Formula C-111) are volatile intermediates, treated with hydride reducing agents such as sodium borohydride, and then acid hydrolyzed to hydroxyl such as compounds of Formulas C-115 and C-116. Obtain the compound.

These various synthetic steps are illustrated in Scheme S-4 below:

A steroid having a C6 hydroxy substituent and a C7 ester substituent can be synthesized according to the procedure illustrated in Scheme S-5 below. In the presence of an acid, an ester at C3 carbonyl (formula C-1) is formed using an ortho ester such as triethyl orthoformate or trimethyl orthoformate trimethyl (such as the compound of formula C-117) to form a 3,5-dienol ether (such as a compound of formula C-117) Such as compounds). Suitable acid is p-toluene sulfonic acid. The enol ether is treated with an oxidizing agent such as meta-chloroperoxybenzoic acid to form a hydroxy compound such as the compound of formula C-118.

Scheme S-6 illustrates the introduction of a double bond at the C1-C2 position of the steroid. This is done by treating the desired steroid (such as Formulas C-1, C-108 and C-114) with a suitable oxidizing agent such as dichlorodicyanobenzoquinone in a suitable solvent (such as dioxane) in the temperature range up to the boiling point. . This procedure can produce C 1 -C 2 unsaturated compounds such as compounds of Formulas C-127, C-128 and C-129.

Scheme S-7 illustrates the introduction of a double bond in the condensed ring. Treatment of steroids (such as compounds of formula C-114) with ortho esters (such as triethyl orthoformate or trimethyl orthoformate) in the presence of an acid catalyst (such as p-toluenesulfonic acid) results in enol ethers (C3 carbonyl). Protected). In the case of compounds of formula C-114, the enol ethers formed are C2-C3 enol ethers (such as compounds of formula C-131) because the C6 position is completely substituted. Seleno derivatives such as compounds of formula C-132 by treatment of enol ethers with strong bases such as lithium diisopropylamide at low temperatures (-78 ° C. to -30 ° C.) followed by selenating agents such as phenyl selenyl chloride Got. In the presence of a base such as pyridine in a solvent such as methylene chloride, for example, oxidizing a seleno derivative with an oxidizing agent such as hydrogen peroxide at room temperature causes removal of the seleno group and introduction of double bonds. Hydrolysis of the enol ether yields a ketone such as the compound of formula C-134.

Scheme S-8 illustrates the synthesis of double bond isomers of compounds of Formula C-1. Treatment of various spiroxane compounds, such as those shown in Scheme S-8, with potassium acetate, trifluoroacetic anhydride and trifluoroacetic acid under conditions similar to those for the synthesis of compounds of formula C-1 would be given by formulas C-121 and C- Obtain 123 compounds.

Scheme S-9 illustrates another method for the synthesis of double bond isomers within the family of such steroids. Scheme S- for the synthesis of compounds such as compounds of formulas C-108 and C-109 starting from preformed enones (such as compounds of formula C-24 described above for their synthesis) that already have a condensed ring The above chemical properties at 3 are used to introduce C6-C7 condensed cyclopropane.

(Wherein X is halogen).

Steroids such as those described in P. Compain, et al., Tetrahedron, 52 (31), 10405-10416 (1996), incorporated herein by reference, in substantially the same manner as discussed above for Scheme S-1. Condensed ring steroids having aromatic A rings can be prepared by treating with trifluoroacetic acid, potassium acetate and trifluoroacetic anhydride.

These novel condensed ring steroids with aromatic A rings and 3-hydroxy substituents are expected to undergo all the typical chemical reactions for phenols. Scheme S-10 illustrates the synthesis of 3-phenolic ethers from such condensed ring steroids. In particular, treatment of these phenolic compounds with bases and halogenated alkyl or alkyl sulfonates is expected to produce the corresponding phenolic acid esters. For example, Feuer and Hooz, In The Chemistry of the Ether Linkage , Patai (Ed.), Interscience: New York, pp. 446-450, 460-468 (1967); And Olson, WT, J. Am. Chem. Soc. , 69, 2451 (1947).

Similarly, Scheme S-11 illustrates the synthesis of 3-phenolic acid esters from condensed ring steroids with aromatic A rings and 3-hydroxy substituents. In particular, treatment of these phenolic compounds with carboxylic anhydride or with carboxylic acid halides is expected to give the corresponding phenolic acid esters. See, eg, March, J., Advanced Organic Chemistry , Wiley: New York, pp. See 346-347 (1985).

Scheme S-12 illustrates the synthesis of 3-phenolic carbonates from condensed ring steroids with aromatic A rings and 3-hydroxy substituents. In particular, treatment of these phenolic compounds with alkyl haloformates is expected to provide the corresponding phenolic carbonates. See, eg, March, J., Advanced Organic Chemistry , Wiley: New York, pp. See 346-347 (1985).

Scheme S-13 illustrates the synthesis of ortho allyl-substituted phenyl derivatives from condensed ring steroids with aromatic A rings and 3-hydroxy substituents. In particular, treatment of these compounds with base and halogenated allyl is expected to give the corresponding allyl phenyl ether. Allyl phenyl ether should provide a mixture of ortho allyl-substituted phenyl derivatives upon thermal rearrangement. See, eg, Shine, H., J. Aromatic Rearrangements; Reaction Mechanism in Organic Chemistry, Monograph 6 , American Elsevier: New York, pp. See 89-120 (1967).

Scheme S-14 illustrates the synthesis of ortho-dialkylated and substituted phenyl derivatives from condensed ring steroids having aromatic A rings and 3-hydroxy substituents. In particular, treatment of these compounds with alcohols and acids is expected to give the corresponding ortho-dialkylated phenyl derivatives. For example, Calcott, WS, J. Am. Chem. Soc. , 61, 1010 (1939).

Scheme S-15 illustrates the allylic oxidation of condensed ring steroids with aromatic A rings and 3-hydroxy substituents. In particular, these compounds can be oxidized at the allyl position by reacting with selenium dioxide and t-butyl hydroperoxide to form the corresponding alcohols. This alcohol is dehydrated to obtain the corresponding olefin. See, eg, Schmuff, NR, J. Org . Chem. , 48, 1404 (1983).

Scheme S-16 illustrates the protection of the 3-carbonyl group and the reduction of the 20-carbonyl group of the novel condensed ring steroids of the present invention. In particular, these compounds can be reacted with trialkylorthoformates and acids to form 3-enol ethers. The 3-enol ether can be reacted with sodium borohydride to reduce the C-20 carbonyl group to the corresponding C-20 alcohol. Treatment of C-20 acools with acid and water deprotects 3-enol ethers to form 3-keto derivatives.

Scheme S-17 illustrates the hydrogenation of olefin bonds in the novel condensed ring steroids of the present invention. In particular, hydrogenation is expected to proceed in stages. Saturate the C-6 and C-7 double bonds first and then saturate the C-8 and C-14 double bonds.

Scheme S-18 illustrates the rearrangement of the protected 11α-hydroxy condensed ring steroids of the present invention. In particular, the 11α-hydroxy group of this compound is first protected with a suitable protecting group such as 2-methoxyethoxymethyl ether (MEM ether). Treatment of protected 11α-hydroxy steroids with alkali metal salts and trihaloalkanoic acid in the presence of acid anhydrides is expected to result in rearrangement of the lactone moieties in these molecules as illustrated below. Removal of the MEM ether with zinc bromide will provide the rearranged alcohol shown below.

Scheme S-19 illustrates the protection of the 3-carbonyl group and alkylation at the 19-position of the novel condensed ring steroids of the invention. In particular, this compound is first converted to 3-alkyl enol ether as illustrated in Scheme S-16. The 3-alkyl enol ether is treated with lithium diisopropylamide (LDA) followed by halogenated alkyl to form a 19-alkyl derivative. Hydrolysis of the 3-alkyl enol ether protecting group yields a 3-keto derivative.

Scheme S-20 illustrates the conversion of estrone methyl ether to the corresponding spirolactone. Rearrangement of lactones to the corresponding condensed ring steroids should occur when the lactones are treated with trihalogenated alkanoic acid under the above reaction conditions, preferably in the presence of alkali metal salts of the alkanoic acid employed. See, for example, Otsubo, K., Tetrahedron Letters , 27 (47), 5763 (1986).

The novel compounds described herein additionally undergo biological conversion processes similar to those described above to allow for other hydroxylated condensed steroids, as well as other novel condensed steroids, such as steroids having 9α, 9β, 11α or 11β-hydroxy substituents. Can be calculated. Then, if desired, the oxidized steroid is oxidized through the removal of hydroxy substituents. Olefin double bonds such as ^9,11 olefin double bonds can be introduced.

In view of the above, it will be seen that several objects of the invention are achieved and other beneficial results obtained.

As various changes can be made in the compositions and methods without departing from the scope of the invention, it is intended that all matter set forth above be interpreted as illustrative and not restrictive.

The following non-limiting examples serve to illustrate various aspects of the invention.

Example X-1A

Methyl 2,3,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3αβ, 11αβ-dimethyl-1,9-dioxo-1H-pentalene [1,6a Preparation of phenanthrene-6α-carboxylate (Compound C-1):

Formula (C-1)

Potassium acetate (6.7 g, 7.1 mmol; Sigma-Aldrich 5128 LG) was added to a clean, dry reactor equipped with a mechanical stirrer, condenser, thermocouple and heating mantle. Trifluoroacetic acid (25.0 mL, 8.1 mol; Sigma-Aldrich 7125MG) and trifluoroacetic anhydride (4.5 mL, 31.0 mmol; Sigma-Aldrich 11828PN) were added to the reactor. This solution was then maintained at a temperature of 25 ° C. to 30 ° C. for 30 minutes.

A preformed TFA / TFA anhydride reagent was prepared in the manner described in Example 36, 7-methyl hydrogen 17-hydroxy-11α- (methylsulfonyl) oxy-3-oxo-17α-pregna-4-ene-7α , 21-dicarboxylate, γ-lactone

Was added to 7 g of.

The resulting mixture was heated at 60 ° C. for 60 minutes and the conversion was checked periodically by TLC and / or HPLC. When the reaction was complete (approximately 60 minutes), the mixture was transferred to a 1 neck flask and concentrated under reduced pressure at 50 ° C. until a thick slurry.

The resulting slurry was diluted with 150 mL of ethyl acetate and 80 mL of water / brine mixture. The phases were then separated and the aqueous layer was reextracted with 80 mL of ethyl acetate. The brine strength was 12% by weight. The combined ethyl acetate solution was washed once with 12 wt% brine (80 mL), then once with 1N NaOH solution (80 mL) and finally with 12 wt% brine (80 mL). The mixture was left for separation and the separated ethyl acetate layer was concentrated and dried under reduced pressure at 45 ° C. using a water inhaler to yield about 3.8 g of crude solid product. HPLC analysis of the crude product revealed that the product contained about 40 area% of compound C-1.

The solid product was then purified by chromatography. Purified by chromatography to give 210 g of methyl 2,3,3a, 4,6,7,9,10,11,11a, 12,13-dodecahydro-3αβ, 11αβ-dimethyl-1,9-dioxo- 1H-pentalene [1,6a-a] phenanthrene-6α-carboxylate (Compound C-1) was produced.

Mass spectrometry data showed that from the high resolution data the molecular weight was 380 and the formula was C ₂₄ H ₂₈ O ₄ . The EI mass spectrum had an M + peak at m / z 380. APCI mass spectra had peaks at m / z 381 (MH) + and m / z 398 (MNH ₄ ) +. Carbon and hydrogen analysis were consistent with the proposed molecular formula.

The IR spectrum had two peaks in the carbonyl absorption region: 1722 cm ⁻¹ and 1667 cm ⁻¹ . ^{Since the 13} C NMR spectra had signals at δ 217.7 due to saturated ketones and at δ 172.7 due to carbomethoxy carbonyl, the 1722 cm ⁻¹ peak was assigned to two carbonyls. The absence of a 1773 cm ^-1 peak in the IR spectrum indicates a loss of the lactone portion.

¹³ C APT and HECTOR NMR data indicated that the following types of carbon were present: 3 carbonyl (σ 217.7, 198.4, 172.2); 4 fully substituted olefinic carbon (σ 166.3, 141.8, 139.3, 121.8); 2 methine olefinic carbons (σ 124.9, 122.0); 3 quaternary aliphatic carbon (σ 61.1, 50.7, 39.7); 1 methine aliphatic carbon (σ 43.3); 8 methylene carbon (σ 46.0, 37.5, 34.1, 33.3, 32.9, 31.9, 23.7, 22.2); And 3 methyl carbon (σ 51.9, 23.6, 23.1).

Example X-1B

(7α, 13R, 17β) -3 ', 4', 5 ', 17-tetrahydro-14-hydroxy-17-methyl-3,5'-dioxo-γ-lactone, cyclopenta [13,17] Preparation of -18-Norlandstar-4,9 (11) -diene-7-carboxylic acid (Compound C-201):

Potassium acetate (8.9 g, 90 mmol), trifluoroacetic acid (150 mL, 1.480 g / mL) and trifluoroacetic anhydride (33 mL, 1.487 g / mL) were 250 mL round bottom reactors with mechanical stirrer, condenser and heating mantle Was added. The resulting solution was stirred at about 25 ° C. to 30 ° C. for about 10 minutes.

15 g (30.0 mmol) of 7-methyl hydrogen 17-hydroxy-11α- (methylsulfonyl) oxy-oxo-17α-pregna-4-ene-7α, 21-dicarboxyl with preformed TFA / TFA anhydride reagent Late, γ-lactone

Was prepared in the manner described in Example 36 above.

The resulting mixture was heated at about 60-70 ° C. for about 1-1.5 hours. The mixture was concentrated at 50 ° C. under reduced pressure to give a thick slurry. The slurry was dissolved in 100 mL of ethyl acetate, washed twice with about 20% water / brine (80 mL each time) and once with 1N sodium hydroxide (80 mL) solution, then 1 with about 20% water / brine (80 mL). Washed twice. The crude product was dried over magnesium sulfate, filtered and concentrated to yield about 18 g of crude wet material.

This material was purified twice by column chromatography to give approximately 3 g of pure (7α, 13R, 17β) -3 ', 4', 5 ', 17-tetrahydro-14-hydroxy-17-methyl-3,5 '-Dioxo-γ-lactone and cyclopenta [13,17] -18-norland star-4,9 (11) -diene-7-carboxylic acid (Compound C-201) were obtained.

Example X-1C

[13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] gona-1,3,5 (10) -triene-5' [2 Preparation of 'H] -one (Compound C-202):

Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid (150 mL, 1.480 g / mL) and trifluoroacetic anhydride (26 mL, 1.487 g / mL) were 250 mL round bottom reactors with mechanical stirrer, condenser and heating mantle Was added. The resulting solution was stirred at about 25 ° C. to 30 ° C. for about 10 minutes.

15 g (43.7 mmol) of 17-hydroxy-3-oxo-17α-pregin-4-ene-21-carboxylic acid, γ-lactone (also known as Aldona; preformed TFA / TFA anhydride reagent; GD Searle & Co .) Was added:

The resulting mixture was heated at 60 ° C. to 70 ° C. for about 1 to 1.5 hours. The reaction mixture was concentrated at 50 ° C. under reduced pressure to give a thick slurry. The slurry was dissolved in 100 ml of ethyl acetate, washed twice with about 20% water / brine solution (80 mL each time) and once with 1N sodium hydroxide solution (80 mL), then about 20% water / brine solution (80 mL) Washed once. The crude product was dried over magnesium sulfate at 50 ° C. under reduced pressure, filtered and concentrated to yield about 20 g of crude wet material.

This material was purified twice by column chromatography to yield approximately 125 g of pure [13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] gona- 1,3,5 (10) -trien-5 '[2'H] -one (Compound C-202) was obtained.

Example X-1D

[13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] gona-1,3,5 (10), 6-tetraene-5' Preparation of [2'H] -one (Compound C-203):

Potassium acetate (6 g, 61.1 mmol), trifluoroacetic acid (150 mL, 1.480 g / mL) and trifluoroacetic anhydride (26 mL, 1.487 g / mL) were 250 mL round bottom reactors with mechanical stirrer, condenser and heating mantle Was added. The resulting solution was stirred at about 25 ° C. to 30 ° C. for about 10 minutes.

15 g (45.9 mmol) of 17-hydroxy-3-oxo prepared from 3-methoxy-3,5,9 (11) -androstaten-17-one (Upjohn) was preformed with TFA / TFA anhydride reagent. -17α-pregine-4,9 (11) -diene-21-carboxylic acid, γ-lactone ( -9,11-also known as Aldona)

Was added. The resulting mixture was heated at about 60 ° C. to 70 ° C. for about 1 to 1.5 hours. The reaction mixture was concentrated at 50 ° C. under reduced pressure to give a thick slurry. The slurry was dissolved in 100 ml of ethyl acetate, washed twice with about 20% water / brine solution (80 mL each time) and once with 1N sodium hydroxide solution (80 mL), then about 20% water / brine solution (80 mL) Washed once. The crude product was dried over magnesium sulfate at 50 ° C. under reduced pressure, filtered and concentrated to yield about 18 g of crude wet material.

This material was purified twice by column chromatography to yield approximately 340 g of pure [13S, 17β] -3 ', 4'-dihydro-3-hydroxy-9,17-dimethylcyclopenta [13,17] gona- 1,3,5 (10), 6-tetraen-5 '[2'H] -one (Compound C-203) was obtained.

Example X-1E

[13S, 17β] -3 ', 4'-dihydro-17-methyl-cyclopenta [13,17] -18-Norlandstar-4,6,8 (14) -triene-3,5' [ Preparation of 2'H] -dione (Compound C-204):

(Compound C-204)

Potassium acetate (8g, 81.5mmol), trifluoroacetic acid (150mL, 1.480g / mL) and trifluoroacetic anhydride (33mL, 1.487g / mL) were 250mL round bottom reactor with mechanical stirrer, condenser and heating mantle Was added. The resulting solution was stirred at about 25 ° C. to 30 ° C. for about 10 minutes.

15 g (44.0 mmol) of 17-hydroxy-3-oxo-17α-pregine-4,6-diene-21-carboxylic acid, γ-lactone (also known as canrenone) of preformed TFA / TFA anhydride reagent; GD Searle & Co.)

Was added.

The resulting mixture was heated at 60 ° C. to 70 ° C. for about 1 to 1.5 hours. The reaction mixture was concentrated at 50 ° C. under reduced pressure to give a thick slurry. The slurry was dissolved in 100 ml of ethyl acetate, washed twice with about 20% water / brine solution (80 mL each time) and once with 1N sodium hydroxide solution (80 mL), then about 20% water / brine solution (80 mL) Washed once. The crude product was dried over magnesium sulfate at 50 ° C. under reduced pressure, filtered and concentrated to yield about 18 g of crude wet material.

This material was purified twice by column chromatography to yield approximately 2.2 g of pure [13S, 17β] -3 ', 4'-dihydro-17-methyl-cyclopenta [13,17] gona-18-Norlandstar -4,6,8 (14) -triene-3,5 '[2'H] -dione (Compound C-204) was obtained.

Example X-2

Preparation of a compound of the formula

(Compound C-105)

Methanesulfonyl chloride (1.1 g, 10 mmol) was added to a stirred cold (0 ° C.) solution of 11α-hydroxykanrenone (3.6 g, 10 mmol) and triethylamine (1.2 g, 12 mmol) in methylene chloride (20 mL). do. The mixture is stirred for 3 hours in cold condition and warmed up to room temperature. Stirring is continued until thin layer chromatography indicates the reaction is complete. The mixture is then diluted with ethyl acetate, extracted with water, 5% aqueous sodium bicarbonate solution and water, and dried over sodium sulfate. The desiccant was filtered and the filtrate was concentrated in vacuo to afford the following crude mesylate C-136 suitable for use in the next step:

(Compound C-136)

Following the procedure described for the synthesis of compound C-1 in Example X-1, a solution of mesylate C-136 (4.3 g, 10 mmol) was added to trifluoroacetic acid (25 mL), trifluoroacetic anhydride (4.5 mL). ) And potassium acetate (6.7 g, 7.1 mmol). The crude product was isolated according to the same procedure as for the compound C-1 in Example X-1 and purified by chromatography on silica gel using ethyl acetate and toluene or a mixture of ethyl acetate and hexane as eluent. do. The product thus obtained is further purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane to yield tetraene C-105.

Example X-3

Preparation of a compound of the formula

(Compound C-110)

Mesylate C-138

11 α, 17-dihydroxy-3-oxo-17-oxo-pregan-4-ene-21-carboxylic acid, γ-lactone (3.6 g, 10 mmol), and triethylamine in methylene chloride (20 mL) ( 1.2 g, 12 moles) and methanesulfonyl chloride (1.1 g, 10 mmol) are used to synthesize and isolate (according to the procedure described in Example X-2 for the synthesis of mesylate C-136). The mesylate C-138 thus isolated is suitable for use in the next step.

According to the procedure described for synthesizing Compound C-1 in Example X-1, a solution of mesylate C-138 (4.4 g, 10 mmol) was added to trifluoroacetic acid (25 mL), trifluoroacetic anhydride (4.5 mL). ) And potassium acetate (6.7 g, 7.1 mmol). The crude product C-110 was isolated according to the same procedure as for the compound C-1 of Example X-1 and subjected to chromatography on silica gel using ethyl acetate and toluene or a mixture of ethyl acetate and hexane as eluent. Purification by The product thus obtained is further purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane.

Example X-4

3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norlandroth-4,8,14-triene-7α- Preparation of Carboxylic Acid (Compound C-101):

A solution of compound C-1 (3.8 g, 10 mmol) and 1N aqueous sodium hydroxide solution (35 mL) in ethanol (60 mL) was refluxed for 8 hours. The reaction is cooled to room temperature, concentrated on a rotary evaporator under vacuum and the remaining aqueous layer is extracted three times with ethyl acetate. The aqueous layer is then acidified with 1N hydrochloric acid solution and extracted three times with ethyl acetate. The combined organic layers are washed with water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated on a rotary evaporator. The remaining crude carboxylic acid C-101 is crystallized by treatment with ethyl acetate and recrystallized from ethyl acetate and hexane or methanol or ethanol and water.

Example X-5

1-Methylethyl 3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-Norlandstar-4,8,14-tree Preparation of En-7α-carboxylate (Compound C-102):

A mixture of a solution of carboxylic acid C-101 (3.7 g, 10 mmol) and isopropyl iodide (3 mL) in sodium bicarbonate (3.5 g) and dimethylformamide (35 mL) is stirred overnight at room temperature. The reaction is poured into water and the aqueous solution is extracted three times with ethyl acetate. The combined organic layers are washed with water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo. The remaining crude isopropyl ester C-102 is crystallized by treatment with ethyl acetate or alcohol, purified by chromatography on silica gel and recrystallized from ethyl acetate and hexane or alcohol or alcohol and water.

Example X-6

Ethyl 3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norlandroth-4,8,14-triene-7α Preparation of carboxylates:

A mixture of a solution of carboxylic acid C-101 (3.7 g, 10 mmol) and ethyl iodide (3 mL) in sodium bicarbonate (3.5 g) and dimethylformamide (35 mL) is stirred overnight at room temperature. The reaction is poured into water and the aqueous solution is extracted three times with ethyl acetate. The combined organic layers are washed with water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo. Residual crude ethyl ester C-103 is crystallized by treatment with ethyl acetate or alcohol, purified by chromatography on silica gel and recrystallized from ethyl acetate and hexane or alcohol or alcohol and water.

Example X-7

Hexyl 3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norlandroth-4,8,14-triene-7α Preparation of Carboxylate (Compound C-104):

A mixture of a solution of carboxylic acid C-101 (3.7 g, 10 mmol) and hexyl iodide (3 mL) in sodium bicarbonate (3.5 g) and dimethylformamide (35 mL) is stirred overnight at room temperature. The reaction is poured into water and the aqueous solution is extracted three times with ethyl acetate. The combined organic layers are washed with water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo. Residual crude n-hexyl ester C-104 is crystallized by treatment with ethyl acetate or alcohol, purified by chromatography on silica gel and recrystallized from ethyl acetate and hexane or alcohol or alcohol and water.

Example X-8

3 ', 4'-dihydro-17-methyl-7α (methylthio) -cyclopenta [13,17] -18-norlandroth-4,8,14-triene-3,5' (2'H Preparation of) -dione (Compound C-106):

A solution of tetraene C-105 (3.2 g, 10 mmol) and piperidine (4 mL) in methanol (40 mL) is cooled to 5 ° C. Pass gas methyl mercaptan until an increase in weight of 7 g is observed. The pressurized container is sealed and kept at room temperature for 20 hours. The solution is poured into ice water, the precipitate is filtered off, washed with water and air dried. Methylthio product C-106 is purified by recrystallization from methanol or ethyl acetate and hexane. See, for example, the procedure described in A. Karim and EA Brown, Steroids, 20 , 41 (1972), incorporated herein by reference.

Example X-9

7α (acetylthio) -3,4'-dihydro-17-methyl-cyclopenta [13,17] -18-norlandroth-4,8,14-triene-3,5 '(2'H) Preparation of Dione (Compound C-107):

A solution of tetraene C-105 (3.2 g, 10 mmol) in thioacetic acid (10 mL) is heated at 85-95 ° C. for 1 hour. Excess thioacetic acid is removed under vacuum and the resulting crude 7α-thioacetate C-107 is purified by recrystallization from methanol or ethyl acetate or a suitable solvent such as ethyl acetate and hexane. See, for example, US Pat. No. 3,013,012, J.A., incorporated herein by reference. Cella and R.C. Tweit, Dec. See the procedure described in 12, 1961.

Example X-10

1,2,4bR (4bR *), 5,5aS *, 7,7aR *, 8,9,11,12bS * -dodecahydro-7a, 12b-dimethyl-10aR * -cyclopropal [ 1 ] pentalene [1,6a-a] phenanthrene-3,10-dione

And

1,2,4bS (4bR *), 5,5aS *, 8,9,11,12,12bR * -dodecahydro-7a, 12b-dimethyl-10aS * -cyclopropal [1] pentalene [1, 6a-a] phenanthrene-3,10-dione (Compound C-109)

Manufacture of:

To a solution of trimethylsulfonium iodide (1 g, 4.6 mmol) in dry dimethyl sulfoxide (20 mL) is added sodium hydride (220 mg of 50% dispersion in mineral oil, 4.6 mmol). The mixture is stirred at room temperature under nitrogen until the release of hydrogen is stopped. Then, a solution of tetraene C-105 (1.12 g, 3.5 mmol) in dimethyl sulfoxide (4 mL) is added and stirring is continued for 4 hours under a nitrogen atmosphere. The reaction mixture is diluted with water, the resulting precipitate is filtered off and air dried. The product is a mixture of 6β, 7β (compound C-108) and 6α, 7α (compound C-109) isomers. These isomers are separated by chromatography on silica gel and each isomer is further purified by recrystallization from solvents such as ethyl acetate and hexane, alcohol, or alcohol and water.

Example X-11

Preparation of a compound of the formula

(Compound C-111)

To the suspension of Enone C-110 (3.2 g, 10 mmol) and anhydrous ethanol (10 mL) in ethyl ortho formate (10 mL) is added p-toluenesulfonic acid monohydrate (0.05 g). The reaction is stirred at room temperature for 30 minutes and quenched by adding a few drops of pyridine. After another 5 minutes of stirring at 0 ° C., the resulting precipitate is filtered, washed with a small amount of methanol and recrystallized from trace amounts of pyridine alcohol or ethyl acetate and hexane to give pure enol ether C-111. .

Alternatively, the reaction may proceed after the addition of pyridine by removing all solvent under vacuum and recrystallizing the residue by addition of a solvent such as ether, ethyl acetate or hexane. Crude C-111 is recrystallized as described above. For example, RM Weier and LM Hofmann, J. Med. See the procedure disclosed in Chem., 20 , 1304-1308 (1977).

Example X-12

Preparation of a compound of the formula

(Compound C-112)

For example, RM Weier and LM Hofmann, J. Med. See Chem., 20 , 1304-1308 (1977).

Vilsmeyer reagent is prepared by adding phosphorus oxychloride (4.59 g, 30 mmol) to dimethylformamide (30 mL) at 0 ° C. After 5 minutes, a solution of enol ether C-111 (3.5 g, 10 mmol) in dimethylformamide (5 mL) is added and the reaction is stirred at 0 ° C. for 2 hours and at room temperature overnight. The reaction is poured into aqueous sodium acetate solution and stirred for 2 hours. The precipitate is filtered and dried to afford crude aldehyde C-112. Purification is carried out by recrystallization from alcohol, alcohol and water or solvents such as ethyl acetate and hexane. Alternatively, crude aldehyde C-112 is isolated from aqueous sodium acetate solution by extraction with a solvent such as ethyl acetate. After drying over sodium sulfate and removing the solvent, the residue is purified by recrystallization or by chromatography on silica gel followed by recrystallization.

Example X-13

Preparation of a compound of the formula

(Compound C-113)

To a stirred cold solution of lithium tri-tert-butoxyaluminum hydride (3.05 g, 12 mmol) in tetrahydrofuran is added aldehyde C-112 (3.78 g, 10 mmol). The reaction is stirred at room temperature for 5 hours, quenched by addition of water and acetic acid, and care is taken to keep the mixture weakly basic. The mixture is concentrated in vacuo and the resulting solid is slurried in ethyl acetate. The solid is filtered off and the filtrate is concentrated in vacuo. The residue is dissolved in a solution of a minimum volume of acetone and water (3: 1) and added to acidified aqueous acetone (pH 1.5-2.0). The acidic reaction mixture is stirred at room temperature for 1 hour and concentrated in vacuo. The resulting crude dienone C-113 is purified by recrystallization from alcohol, alcohol and water or solvents such as ethyl acetate and hexane. Alternatively, crude dienone C-113 is purified by chromatography on silica gel and then recrystallized. For example, RM Weier and LM Hofmann, J. Med. See the procedure described in Chem., 20 , 1304-1308 (1977).

Example X-14

2 ', 3', 3'aα, 4 ', 6', 10 ', 11', 11'aα, 12 ', 13'-decahydro-3'aR, 3'a, 11'a-dimethyl-13 Preparation of 'aR * -spiro [cyclopropane-1,7' (9'H)-[1H] pentalene [1,6a-a] phenanthrene-1'9'-dione:

(Compound C-114)

To a solution of dienone C-113 (1.17 g, 0.05 mmol) in tetrahydrofuran (30 mL) is added a solution of diazomethane (0.2 g, 0.07 mmol) in ether (7 mL). The resulting reaction solution is stored for several days at room temperature. Acetic acid is then added to remove excess diazomethane and the reaction is concentrated in vacuo. The residue, intermediate pyrazoline, is crystallized in a solvent such as acetone, hexane, ethyl acetate or ethanol and then recrystallized. This material is converted to spirocyclopropane C-114. Thus, solid C-114 is heated at 190 ° C. under vacuum and the resulting solid is recrystallized from alcohol, alcohol and water, acetone and water or ethyl acetate and hexane. Alternatively, a solution of pyrazoline in acetone is treated with boron trifluoride etherate at room temperature for about 1 hour. Water is added, the resulting precipitate is filtered off and air dried. Purification is performed by recrystallization. For example, F.B., incorporated herein by reference. Colton and R.T. See the procedure described in Nicholson, US Pat. No. 3,499,891, March 10, (1970).

Example X-15

Preparation of a compound of the formula

(Compound C-115)

And

(Compound C-116)

To a solution of enol ether C-111 (3.2 g, 10 mmol) in methanol (20 mL) is added sodium borohydride (38 mg). The reaction is stirred at rt for 3 h and treated with 1N hydrochloric acid for 30 min. The reaction is further diluted with water, the precipitate is filtered off, washed with water and dried. The product consisting of a mixture of two epimeric alcohols (Compound C-115 and Compound C-116) is separated by chromatography on silica gel. Purified alcohols C-115 and C-116 are recrystallized from alcohol, alcohol and water, ethyl acetate and hexane and solvents such as acetone and hexane, respectively. Alternatively, the diluted reaction mixture is extracted with ethyl acetate, the combined organic layers are dried over sodium sulfate, and the crude product thus obtained is chromatographed as described above to give separated alcohols C-115 and C-116.

Example X-16

Preparation of a compound of the formula

(Compound C-117)

Compound using ethyl orthoformate (3.2 g, 10 mmol), ethanol (10 mL) and p-toluenesulfonic acid monohydrate (0.05 g) according to the procedure described for the synthesis of compound C-111 described in Example X-11 Convert C-1 (3.8 g, 10 mmol) to enol ether C-117.

Example X-17

3 ', 4', 5 ', 17-tetrahydro-6α-hydroxy-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-Norlandstar-4,8,14- Preparation of Triene-7α-carboxylate (Compound C-118):

(Compound C-118)

A solution of 57% m-chloroperoxybenzoic acid (3.64 g) in 10% aqueous dioxane (20 mL) is semi-neutralized with 1N sodium hydroxide solution. The solution is cooled to 0 ° C. and added in part to a stirred cold solution of enol ether C-117 (4.1 g, 10 mmol) in 10% aqueous dioxane (20 mL). The reaction is stirred overnight at room temperature, poured into ice water and extracted with methylene chloride or ethyl acetate. The combined organic layers are dried over sodium sulfate. The solvent is evaporated to afford crude hydroxy ester C-118, which is purified by chromatography on silica gel. For example, RM Weier and LM Hofmann, J. Med. See the procedure described in Chem., 20 , 1304-1308 (1977).

Example X-18

Preparation of a compound of the formula

(Compound C-117)

Lithium dimethyl cuprate is prepared by adding methyllithium (19 mL of a 1.6 M solution in ether, 30 mmol) to a stirred suspension of cuprous iodide (2.86 g, 15 mmol) in ether (30 mL) at 0 ° C. under an argon inert atmosphere. After stirring under cold for 15 minutes, a solution of tetraene C-105 (1.6 g, 15 mmol) in tetrahydrofuran (25 mL) was added dropwise over 25 minutes. The reaction is reacted for another 30 minutes and poured into saturated ammonium chloride solution with vigorous stirring. The aqueous mixture is extracted with ethyl acetate or methylene chloride. The combined organic layers are washed with aqueous ammonium chloride solution and water and dried over sodium sulfate. The solvent is removed in vacuo and the residue is dissolved in ethyl acetate or methylene chloride and treated with p-toluenesulfonic acid (100 mg) in a steam bath for 30 minutes to 1 hour. The organic solution is washed with water and dried over sodium sulfate. The solvent is removed in vacuo to afford crude Enone C-119. Purification is carried out by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane. Alternatively, crude Enone C-119 is chromatographed on silica gel and then recrystallized. See, for example, the procedure described in JK Grunwell et al., Steroids, 27 , 759-771 (1976), incorporated herein by reference.

Example X-19

Preparation of a compound of the formula: (Compound C-121)

Ester C-120 (4.00 g, 10 mmol)

(Compound C-120)

(Prepared according to the procedures described in RM Weier and LM Hofmann, J. Med. Chem., 18, 817 (1975), incorporated herein by reference), the synthesis of Compound C-1 in Example X-1. React with trifluoroacetic acid (25 mL), trifluoroacetic anhydride (4.5 mL) and potassium acetate (6.7 g, 7.1 mmol) following the procedure described for. The crude product C-121 was isolated according to the same procedure as for the compound C-1 of Example X-1 and subjected to chromatography on silica gel using ethyl acetate and toluene or a mixture of ethyl acetate and hexane as eluent. The product thus obtained is further purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane.

Example X-20

Preparation of a compound of the formula

(Compound C-123)

Enon C-122 (3.68 g, 10 mmol)

(Compound C-122)

(Prepared according to the procedure described in FB Colton and RT Nicholson, US Patent 3,499,891, March 10, (1970), incorporated herein by reference), for the synthesis of Compound C-1 in Example X-1. React with potassium acetate (6.7 g, 7.1 mmol), trifluoroacetic acid (25 mL) and trifluoroacetic anhydride (4.5 mL) following the procedure. The crude product is isolated as described in the above reference and purified by chromatography on silica gel using ethyl acetate and hexane or a mixture of ethyl acetate and toluene as eluent. Purified C-123 is further purified by recrystallization from alcohol, alcohol and water, or ethyl acetate and hexane.

Example X-21

Preparation of a compound of the formula

(Compound C-125)

And

(Compound C-126)

Compounds C-125 and C-126 are synthesized according to the procedures used above for the synthesis of compounds C-108 and C-1. Thus, sodium hydride (220 mg of 50% dispersion in mineral oil, 4.6 mmol) is added to a solution of trimethylsulfonium iodide (1 g, 4.6 mmol) in dry dimethyl sulfoxide (20 mL). The mixture is stirred at room temperature under nitrogen until the release of hydrogen is stopped.

Then, trienone C-124 (1.14 g, 3.5 mmol) in dimethyl sulfoxide (4 mL)

(Compound C-124)

Solution is added and stirring is continued for 4 hours under a nitrogen atmosphere. Trienone C-124 is prepared according to the procedure described in Example X-2 for the preparation of tetraene C-105 by using canrenone instead of 11α-hydroxy canrenone as starting material. The reaction mixture is diluted with water and the resulting precipitate is filtered and air dried. The product is a mixture of 6β, 7β (compound C-125) and 6α, 7α (compound C-126) isomers. These isomers are separated by chromatography on silica gel and the individual isomers are further purified by recrystallization from solvents such as ethyl acetate and hexane, alcohol or alcohol and water.

Example X-22

3 ', 4', 5 ', 17-tetrahydro-17β-methyl-3,5'-dioxocyclopenta [13R, 17] -18-norlandroth-1,4,8,14-tetraene- Preparation of 7α-carboxylate (Compound C-127):

(Compound C-127)

R.M., incorporated herein by reference. Weier and L.M. Hofmann, J. Med. Dienone C-127 is synthesized according to the procedure described in Chem., 18, 817 (1975). Thus, a solution of compound C-1 (3.8 g, 10 mmol) and dichlorodicyanobenzoquinone (2.72 g, 12 mmol) in dioxane (80 mL) was refluxed with stirring for 24 h. The reaction mixture is concentrated in vacuo, the residue is degraded with methylene chloride, filtered and the filtrate is washed with 2% sodium sulfite, 5% sodium hydroxide and saturated sodium chloride solution and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo. The crude product Dienone C-127 is purified by chromatography on silica gel using a mixture of ethyl acetate and toluene as eluent and the product 27 thus isolated is further purified by recrystallization from alcohol.

Example X-23

2 ', 3', 3'aα, 4 ', 6'11'aα, 12', 13'-octahydro-3'aR, 3'a, 11'a-dimethyl-13'aR * -spiro [cyclo Preparation of Propane-1,7 '(9'H)-[1H] pentalene, [1,6a-a] phenanthrene] 1'9'-dione (Compound C-128):

(Compound C-128)

Using the procedures and operations described in Example X-22 for the synthesis of Dienone C-127, Enone C-114 (3.48 g, 10 mmol) was converted to dichlorodicyanobenzoquinone (2.72 g, 12 mmol) is converted to dienone C-128.

Example X-24

4bR (4bR *), 5,5aS *, 7,7aR *, 8,9,11,12,12bS * -decahydro-7A, 12b-dimethyl-10R * -cyclopropa ( 1 ) pentalene [1, Preparation of 6a-a] phenanthrene-3,10-dione (Compound C-129):

(Compound C-129)

Using the procedures and operations described in Example X-22 for the synthesis of Dienone C-127, Enone C-108 (3.34 g, 10 mmol) was converted to dichlorodicyanobenzoquinone (2.72 g, 12 mmol) is converted to Dienone C-129.

Example X-25

Preparation of a compound of formula

(Compound C-130)

Isobutyl chloroformate (1.36 g, 10 mmol) in 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). Add). The reaction is stirred at 0 ° C. for 20 minutes, filtered and the filtrate is concentrated in vacuo. The residue is mixed anhydride and is suitable for use in the next step.

Bubbles are generated with gaseous dimethylamine in a cold (0 ° C.) solution of mixed anhydride (4.6 g, 10 mmol) in tetrahydrofuran (40 mL) in a pressurized vessel. After 15 minutes, the pressure vessel is sealed and the reaction is left at room temperature for 24 hours. The reaction is warmed to 40 ° C. for 30 minutes and cooled back to 0 ° C. After warming up to room temperature, the vessel is vented to atmosphere and excess dimethylamine is evaporated. The reaction is concentrated in vacuo and the residue is dissolved in ethyl acetate and extracted with 1N sodium hydroxide solution and water. After drying over sodium sulfate, the organic layer is stripped and the residue is purified by chromatography on silica gel using a mixture of ethyl acetate and toluene as eluent. The amide C-130 so isolated is further purified by recrystallization from alcohol, alcohol and water or ethyl acetate and hexane.

Example X-26

Preparation of a compound of the formula

(Compound C-131)

To a suspension of Enone C-114 (3.5 g, 10 mmol) in ethyl ortho formate (10 mL) and anhydrous ethanol (10 mL) is added p-toluenesulfonic acid monohydrate (0.05 g). The reaction is stirred at room temperature for 30 minutes and quenched by adding a few drops of pyridine. After stirring for 5 minutes at 0 ° C., the resulting precipitate is filtered, washed with a small amount of methanol and recrystallized from alcohol or ethyl acetate and hexane containing trace amounts of pyridine to give pure enol ether C-131.

Alternatively, the reaction may be carried out after addition of pyridine by removing all solvent under vacuum and crystallizing the residue by adding a solvent such as hexane, ether, ethyl acetate or hexane. Crude enol ether C-131 is recrystallized as described above.

Example X-27

Preparation of a compound of the formula

(Compound C-132)

To a cold (-78 ° C) solution (10 mL of 1.0 M solution in tetrahydrofuran) of lithium bis (trimethylsilyl) amide in 20 minutes add a solution of enol ether C-131 (3.8 g, 10 mmol) in tetrahydrofuran (20 mL). It is dripped over. The reaction is stirred at −78 ° C. for 10 minutes and a solution of phenylselenyl chloride (1.9 g, 10 mmol) in tetrahydrofuran (5 mL) is added. The reaction is stirred for 5 minutes and quenched by addition of 1% sodium hydrogen sulfate solution. The reaction is further diluted with water and extracted with ethyl acetate. The combined organic layers are washed with 5% aqueous sodium bicarbonate solution, washed with water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo. The residue is purified by chromatography on silica gel using ethyl acetate and toluene or a mixture of ethyl acetate and hexane as eluent to afford pure selenide C-132.

Example X-28

Preparation of a compound of the formula

(Compound C-133)

To a cold (0 ° C) solution of selenide C-132 (g, 10 mmol) and pyridine (1.61 mL, 20 mmol) in methylene chloride (40 mL) was added a solution of hydrogen peroxide (30 g solution 3.1 g, 27 mmol) in water (3 mL). Add slowly. Keep the temperature below 30-35 ° C. After the exotherm has settled, the ice bath is removed and the reaction is stirred vigorously for 15 minutes at room temperature. The reaction is diluted with methylene chloride, washed with 5% sodium bicarbonate solution and water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo. Purification by chromatography on silica gel using ethyl acetate and toluene or a mixture of ethyl acetate and hexane as eluent gives pure unsaturated ketone C-133, which is further purified by recrystallization from ethyl acetate and hexane or alcohol.

X-29 on implementation

Preparation of a compound of the formula

(Compound C-134)

A solution of ketone C-133 (2 g) in acetone (15 mL) is treated with 1N hydrochloric acid (4 mL) at room temperature for 1 hour. The reaction is concentrated in vacuo. The residue is diluted with water and extracted with ethyl acetate. The combined organic layers are washed with 5% sodium bicarbonate solution and water and dried over sodium sulfate. The desiccant is filtered and the filtrate is concentrated in vacuo to afford crude ketone C-134. The crude product is purified by chromatography on silica gel using a mixture of ethyl acetate and toluene as eluent to afford pure ketone C-134, which is further purified by recrystallization from ethyl acetate and hexane or alcohol.

Claims (152)

A process for preparing 3-keto-7α-alkoxycarbonyl substituted Δ-4,5-steroid, comprising reacting an alkylating agent with a 4,5-dihydro-5,7-lactone steroid substrate in the presence of a base, Lactone substrates are substituted with keto or dialkoxy at 3-carbon, with (Formula XXX) Wherein C (5) represents 5-carbon of the steroid structure of the substrate and C (7) represents 7-carbon of the steroid structure of the substrate). The method of claim 1 wherein the steroid substrate comprises a saturated or unsaturated nucleus structure of the formula XLI or XLII: Formula XLI Formula XLII Where -AA- represents -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ^5- ; R ³ , R ⁴ and R ⁵ are groups consisting of hydrogen, halogen, hydroxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy Independently from; -BB- is a -CHR ⁶ -CHR ⁷ -group or the next group in an alpha- or beta- orientation Formula III Wherein R ⁶ and R ⁷ are hydrogen, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryloxy groups Selected from the group consisting of); R ⁸⁰ and R ⁹⁰ are independently selected from R ⁸ and R ⁹ , respectively, or R ⁸⁰ and R ⁹⁰ together form a keto; R ⁸ and R ⁹ are independently hydrogen, hydroxy, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, acyloxyalkyl, cyano and aryl Selected from the group consisting of oxy groups, or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ is a 5-membered cyclic D ring To constitute a carbocyclic or heterocyclic ring structure fused to; -E-E- is: Formula XLIII , Formula XLIV , Formula XLV , (Formula XLVI) And Formula XLVII (Wherein R ²¹ , R ²² and R ²³ are independently selected from hydrogen, alkyl, halo, nitro and cyano and R ²⁴ is selected from hydrogen and C ₁ -C ₄ alkyl; R ¹⁸ is C ₁ -C ₄ alkyl or R ¹⁸ O— group together forming an O, O-oxyalkylene bridge) Is selected from. The compound of claim 2, wherein the steroid substrate and the steroid product are in the 17-position (Formula XXXIII) A method characterized in that it is substituted with a spirolactone substituent corresponding to. The method of claim 1 wherein the steroid substrate and the steroid product are substituted with keto substituents at the 17-position. The method of claim 1 wherein the lactone substrate comprises a 3-dialkoxy substituent. The method of claim 1 wherein the 5,7-lactone is reacted with halogenated alkyl in the presence of a base. delete delete Formula II Formula II (Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group; R ¹ represents an alpha-oriented C ₁ -C ₄ alkoxycarbonyl or hydroxycarbonyl radical; R ³ , R ⁴ and R ⁵ are independently hydrogen, halo, hydroxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy Selected from the group consisting of; -BB- is a -CHR ⁶ -CHR ⁷ -group or the next group in an alpha- or beta- orientation Formula III Wherein R ⁶ and R ⁷ are independently hydrogen, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryl Selected from the group consisting of oxy groups); R ⁸ and R ⁹ are independently hydrogen, hydroxy, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, acyloxyalkyl, cyano and aryl Selected from the group consisting of oxy groups, or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ is a 5-membered cyclic D ring A method for preparing a compound corresponding to a carbocyclic or heterocyclic ring structure fused to Alkylating the compound of formula EI by hydrolysis and then reacting with an alkylating agent in the presence of a base, The compound of formula EI has the structure Formula EI Wherein, -AA-, R ³ , R ⁸ , R ⁹ and -BB- are as defined above and R ¹⁷ is C ₁ to C ₄ alkyl. Chemical Formula IIC Formula IIC (Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group; R ¹ represents an alpha-oriented C ₁ -C ₄ alkoxycarbonyl or hydroxycarbonyl radical; R ³ , R ⁴ and R ⁵ are independently hydrogen, halo, hydroxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy Selected from the group consisting of; -BB- is a -CHR ⁶ -CHR ⁷ -group or the next group in an alpha- or beta- orientation Formula III Wherein R ⁶ and R ⁷ are independently hydrogen, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryl Is selected from the group consisting of an oxy group). Alkylating the compound of formula E by hydrolysis, followed by reaction with an alkylating agent in the presence of a base, The compound of formula E has the structure Formula E Wherein -AA-, -BB- and R ³ are as defined above and R ¹⁷ is C ₁ to C ₄ alkyl. delete delete delete delete Formula I Formula I (Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group; R ³ , R ⁴ and R ⁵ are independently hydrogen, halo, hydroxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy Selected from the group consisting of; R ²⁶ is C ₁ to C ₄ alkyl; -BB- is a -CHR ⁶ -CHR ⁷ -group or the next group in an alpha- or beta- orientation Formula III Wherein R ⁶ and R ⁷ are independently hydrogen, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryl Selected from the group consisting of oxy groups); R ⁸⁰ and R ⁹⁰ are independently selected from R ⁸ and R ⁹ , respectively, or R ⁸⁰ and R ⁹⁰ together form a keto; R ⁸ and R ⁹ are independently hydrogen, hydroxy, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, acyloxyalkyl, cyano and aryl Selected from the group consisting of oxy groups, or R ⁸ and R ⁹ together form a carbocyclic or heterocyclic ring structure, or R ⁸ or R ⁹ together with R ⁶ or R ⁷ is a 5-membered cyclic D ring A method for preparing a compound corresponding to a carbocyclic or heterocyclic ring structure fused to Alkylating the compound of Formula A211 by reacting with an alkylating agent in the presence of a base, A compound of formula A211 has the structure Formula A211 (Wherein -AA-, -BB-, R ³ , R ⁸⁰ and R ⁹⁰ are as defined above). Formula IE (Formula IE) (Wherein -AA- represents a -CHR ⁴ -CHR ⁵ -or -CR ⁴ = CR ⁵ -group; R ³ , R ⁴ and R ⁵ are independently hydrogen, halo, hydroxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, cyano and aryloxy Selected from the group consisting of; R ²⁶ is C ₁ to C ₄ alkyl; -BB- is a -CHR ⁶ -CHR ⁷ -group or the next group in an alpha- or beta- orientation Formula III Wherein R ⁶ and R ⁷ are independently hydrogen, halo, C ₁ -C ₄ alkoxy, acyl, hydroxyalkyl, alkoxyalkyl, hydroxycarbonyl, alkyl, alkoxycarbonyl, acyloxyalkyl, cyano and aryl Is selected from the group consisting of an oxy group). Alkylating the compound of Formula 211 by reacting with an alkylating agent in the presence of a base, The compound of formula 211 is Formula 211 (Wherein -AA-, -BB- and R ³ are as defined above). delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete