Process for the preparation of intermediates for 11-beta,16-alpha,17-alpha,21-tetrahydroxypregna-3,20-dione 16-alpha,17-alpha-acetals and 21-esters thereof

Abstract: The invention relates to the preparation of a compound of the formula or a stereoisomeric compound thereof, in which formula the the l,2-position is saturated or is a double bond; X₁ is selected from hydrogen, fluorine, chlorine and bromine X₂ is selected from hydrogen, fluorine, chlorine and bromine R₂ is selected from straight and branched hydrocarbon chains having l-l0 carbon atoms and R₇ is hydrogen or an acyl group with l-l0 carbon atoms arranged in a straight or branched chain, characterized by reaction of a steroid compound of the formula with an aldehyde compound of the formula wherein X₁, X₂, R₂, R₇ and have the meanings given above, in the presence of an acid catalyst. The products of the process are useful intermediates in the preparation of novel glucocorticosteroids which possess high anti-inflammatory potency at the place of application and low glucocorticord systemic potency.

Description: The present invention relates to a process for the preparation of intermediates for producing pharmacologically active compounds that are described and claimed in European Patent Specification No. 0197018. It is known that certain glucocorticosteroids (GCS) can be used for local therapy of inflammatory, allergic or immunologic diseases in respiratory airways (e.g. asthma, rhinitis), in skin (eczema, psoriasis) or in bowel (ulcerative colitis, Morbus Crohn). With such local glucocorticoid therapy, clinical advantages over general therapy (with e.g. glucocorticoid tablets) are obtained, especially regarding reduction of the unwanted glucocorticoid effects outside the diseased area. To reach such clinical advantages, in e.g. severe respiratory airway disease, GCS must have a suitable pharmacological profile. They should have high intrinsic glucocorticoid activity at the application site but also a rapid inactivation by e.g. hydrolysis in the target organ or after uptake into the general circulation. Since binding of GCS to the glucocorticoid receptor is a pre-requisite for their anti-inflammatory and allergic effects to occur, the ability of steroids to bind to their receptor(s) can be used as an adequate method for determining the biological activity of GCS. A direct correlation between the affinity of GCSs to the receptor and their antiinflammatory effects has been shown using ear edema test in the rat. [Correlation between chemical structure, receptor binding, and biological activity of some novel, highly active, 16α,17α-acetalsubstituted glucocorticoids. E. Dahlberg, A. Thalén, R. Brattsand, J-Å Gustafsson, U. Johansson, K. Roempke, and T. Saartok, Mol. Pharmacol. 25 (1984), 70.] European Patent Specification No. 0197018, which is the parent of the present divisional specification, discloses that certain 3-oxoandrosta-1,4-diene-17β-carboxylic acid esters possess high binding affinity to the glucocorticosteroid receptor. In particular, the compounds disclosed in European Patent Specification No. 0197018, which can be used for the treatment and control of inflammatory conditions, are characterized by the formula: wherein the 1,2-position is saturated or is a double bond; X₁ is selected from hydrogen, fluorine, chlorine and bromine; X₂ is selected from hydrogen, fluorine, chlorine and bromine; R₁ is selected from hydrogen and a straight or branched hydrocarbon chain having 1-4 carbon atoms; R₂ is selected from hydrogen and straight or branched hydorcarbon chains having 1-10 carbon atoms; and R₃ is selected from Y is O or S; R₄ is selected from hydrogen, straight or branched hydrocarbon chains having 1-10 carbon atoms and phenyl; R₅ is selected from hydrogen and methyl; and R₆ is selected from hydrogen, straight or branched, saturated or unsaturated hydrocarbon chains having 1-10 carbon atoms, an alkyl group substituted by at least one halogen atom, a heterocyclic ring system containing 3-10 atoms in the ring system, (m= 0,1,2; n= 2,3,4,5,6), and phenyl or benzyl groups which are unsubstituted or substituted by one or more alkyl, nitro, carboxy, alkoxy, halogen, cyano, carbalkoxy or trifluoromethyl group(s), provided that when R₂ is hydrogen R₁ is a straight or branched hydrocarbon chain having 1-4 carbon atoms. SE-A-378109 discloses an alternative method for the production of the here described steroids starting from the corresponding 16,17-diol. The present invention is directed to a process for the preparation of a compound of the formula D : or a stereoisomeric compound thereof, in which formula: the 1,2-position is saturated or is a double bond; X₁ is selected from hydrogen, fluorine, chlorine and bromine; X₂ is selected from hydrogen, fluorine, chlorine and bromine; R₂ is selected from straight and branched hydrocarbon chains having 1-10 carbon atoms; and R₇ is hydrogen or an acyl group with 1-10 carbon atoms arranged in a straight or branched chain, characterized by reaction of a compound of the formula F : with a compound of the formula wherein X₁, X₂, R₂, R₇ and --- have the meaning given above, in the presence of perchloric acid. European Patent Specification No. 0197018 discloses that the individual stereoisomeric components present in a mixture of a steroid having the above formula (I) can be elucidated in the following way: The individual stereoisomeric components present, in a mixture of steroid 17β-carboxylic acid esters having the formulas where St is the steroid moiety, can be elucidated in the following way In diasteroisomers like II, III, VI, VII, VIII and IX, the configuration differs only at one out of several asymmetric carbon atoms. Such diastereoisomers are denoted epimers. Alkyl in the definitions above is a straight or branched hydrocarbon chain with 1-5 carbon atoms, preferably 1-4 C. Alkoxy in the definition above is a group -O-alkyl wherein the alkyl moiety has the given definition. Halogen in the definition above is preferably a chlorine, bromine or fluorine atom. Carbalkoxy in the definition above is a group -COO-alkyl wherein the alkyl moiety has the above given definition. Heterocyclic ring system is a ring system containing as hetero atoms N, O or S. Preferred systems are pyrryl, pyrridyl, pyrimidyl, pyrazinyl, furyl, pyranyl, benzofuranyl, indolyl and thienyl. Preferred compounds described and claimed in European Patent Specification No. 0197018 are: l′Ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(l-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17β-carboxylate, the epimeric mixture A + B and epimer B. l′-isopropoxycarbonyloxyethyl 9α-fluoro-11β-hydroxy-16α,17α-[(l-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17β-carboxylate, epimer B. l′propoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(l-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17β-carboxylate, epimer B. l′-isopropoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(l-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17β-carboxylate, epimeric mixture A + B and epimer B. l′-Acetoxyethyl (2OR)-9α-fluoro-11β-hydroxy-16α,17α-propylmethylenedioxyandrosta-1,4-diene-3-one-17β-carboxylate, epimer B. l′-Ethoxycarbonyloxyethyl (22R)-9α-fluoro-11β-hydroxy-16α,17α-propylmethylenedioxyandrosta-1,4-diene-3-one-17β-carboxylate, epimer B. l′-isopropoxycarbonyloxyethyl (2OR)-9α-fluoro-11β-hydroxy-16α,17α-propylmethylenedioxyandrosta-1,4-diene-3-one-17β-carboxylate, epimer B. l′-Ethoxycarbonyloxyethyl (2OR)-6α,9α-fluoro-11β-hydroxy-16α,17α-propylmethylenedioxyandrosta-1,4-diene-3-one-17β-carboxylate, epimeric mixture A + B and epimer B. Methods of Preparation The compounds of formula I of European Patent Specification No. 0197018 are prepared by the oxidation of a compound of the formulas X, XI and XII to the corresponding 17β-carboxylic acid: wherein the solid and broken lines between C-1 and C-2 represent a single or double bond, X₁, X₂, R₁ and R₂ have the meaning given above for formula I, and R₇ is hydrogen or an acyl group with 1-10 carbon atoms arranged in a straight or branched chain. The 17β-carboxylic acids then are esterified to give compounds characterized by the formula I-IX, wherein --- X₁, X₂, R₁, R₂ and R₃ have the meaning given above for formula I. The process of this invention to convert a compound of formulas X, XI or XII to the corresponding 17-carboxylic acids is carried out in a suitable oxygenated hydrocarbon solvent such as a lower alkanol. Methanol and ethanol are preferred, particularly the former. The reaction medium is made slightly alkaline by the addition of a suitable weak inorganic base such as an alkali metal carbonate, for example sodium, lithium or potassium carbonate. The latter is perferred. The conversion of a compound of formula X, XI or XII to a 17β-carboxylic acid of formula I, II or III (R₃=H) takes place at ambient temperatures, i.e. 20-25°C. The presence of oxygen is necessary for the reaction. Oxygen can be supplied by bubbling a stream of air or oxygen into the reaction mixture. The oxidative degradation of the 17β side-chain of compounds of formula X, XI and XII to the corresponding 17β carboxylic acids can also be carried out with periodic acid, sodium hypobromate or with sodium bismuthate. The reaction is performed in a mixture of water and a suitable oxygenated hydrocarbon solvent such as a lower ether. Dioxane and tetrahydrofurane are preferred, particularly the former. The parent 17β-carboxylic acids of compounds of formula I, II and III (R₃=H) may be esterified in known manner to provide 17βcarboxylate esters according to the invention. For example, the 17β-carboxylic acid may be reacted with an appropriate alcohol and a carbodiimide, e.g. dicyclohexylcarbodiimide, in a suitable solvent such as diethylether, tetrahydrofurane, methylene chloride or pyridine advantageously at a temperature of 25-100°C. Alternatively, a salt of the 17β-carboxylic acid with an alkali metal, e.g. lithium, sodium or potassium, a salt of a quaternary ammonium compound, such as a salt of triethyl-or tributylamine, or tetrabutylammonium, may be reacted with an appropriate alkylating agent, for example an acyloxyalkylhalide or haloalkyl alkylcarbonate preferably in a polar solvent medium such as acetone, methylenechloride or chloroform, conveniently at a temperature in the range of 25-100°C. The reaction may also be performed in the presence of a crown ether. The crude steroid ester derivates formed are after isolation purified by chromatography on a suitable material, for instance cross-linked dextran gels of Sephadex® LH-type with suitable solvents as eluants, e.g. halogenated hydrocarbons, ethers, esters such as ethyl acetate or acetonitrile. The individual epimers, which are formed at the acetalisation to the 16α,17α-hydroxy groups or at the esterification of the 17β-carboxylic acids, possess practically identical solubility characteristics. Accordingly, they have turned out to be impossible to separate and isolate from the epimeric mixture by conventional method for resolution of stereoisomers, e.g. fractionated crystallization. In order to obtain the individual epimers separately the stereoisomeric mixtures according to the formulas I, IV an V above are subjected to column chromatograhy, thus separating the epimers II, III, VI, VII, VIII and IX in view of different mobility on the stationary phase. The chromatography may be carried out for instance on cross-linked dextran gels of the type Sephadex® LH, e.g. Sephadex® LH-20 in combination with a suitable organic solvent as eluting agent. Sephadex® LH-20, prepared by Pharmacia Fine Chemicals AB, Uppsala, Sweden, is a beadformed hydroxypropylated dextran gel wherein the dextran chains are cross-linked to give a three-dimensional polysaccharide network. As eluting agent, halogenated hydrocarbons e.g. chloroform or a mixture of heptane-chloroform-ethanol in the proportions 0-50:50-100:10-1 has successfully been used, preferably a 20:20:1 mixture. Compounds X, XI and XII are used as starting materials for compounds of formula I. They are used by transacetalisation of the corresponding 16α,17α-acetonides wherein the solid and broken lines between C₁ and C₂ represent a single or double bond and X₁, X₂ and R₇ have the meaning given above with an aldehyde of the formula wherein R₂ has the meaning given above. The aldehyde is preferably acetaldehyde, propanal, butanal, isobutanal, pentanal, 3-methylbutanal, 2,2-dimethylpropanal, hexanal, heptanal, octanal, nonanal and dodecanal. The reaction is carried out by adding the steroid to a solution of the aldehyde together with perchloric acid as catalyst in an ether, preferably dioxane or tetrahydrofurane, a halogenated hydrocarbon, preferably methylene chloride or chloroform, an aromatic hydrocarbon, preferably toluene, an alicyclic hydrocarbon, preferably cyclohexane or an aliphatic hydrocarbon, preferably heptane or isooctane, under the latter conditions eliminating the chromatographic step for preparation of the epimers III and XII. Pharmaceutical Preparations The compounds of formula I may be used for different modes of local administration dependent on the site of inflammation, e.g. percutaneously, parenterally or for local administration in the respiratory tract by inhalation. An important aim of the formulation design is to reach optimal bioavailability of the active steroid ingredient. For percutaneous formulations this is advantageously achieved if the steroid is dissolved with a high thermodynamic activity in the vehicle. This is attained by using a suitable system of solvents comprising suitable glycols, such as propylene glycol or 1,3-butandiol either as such or in combination with water. It is also possible to dissolve the steroid either completely or partially in a lipophilic phase with the aid of a surfactant as a solubilizer. The percutaneous compositions can be an ointment, an oil in water cream, a water in oil cream or a lotion. In the emulsion vehicles the system comprising the dissolved active component can make up the disperse phase as well as the contionuous one. The steroid can also exist in the above compositions as a micronized, solid substance. Pressurized aerosols for steroids are intended for oral or nasal inhalation. The aerosol system is designed in such a way that each delivered dose contains 10-1000 µg, preferably 20-250 µg of the active steroid. The most active steroids are administered in the lower part of the dose range. The micronized steroid consists of particles substantially smaller than 5 µm, which are suspended in a propellent mixture with the assistance of a dispersant, such as sorbitan trioleate, oleic acid, lecithin or sodium salt of dioctylsulphosuccinic acid. Working Examples The invention will be further illustrated by the following non-limitative examples. In the examples a flow-rate of 2.5 ml/cm²· h⁻¹ is used at the preparative chromatographic runs. Molecular weights are in all examples determined with electron impact mass spectrometry and the melting points on a Leitz Wetzlar hot stage microscope. All HPLC analyses (HPLC = High Performance Liquid Chromatography) were performed on a Waters µBondapak C₁₈ column (300x3.9 mm internal diameter) with a flow-rate of 1.0 ml/min and with ethanol-water in ratios between 50:50 and 60:40 as mobile phase, if not otherwise stated. Example 1 . This example sets forth a process for preparing (22RS)-, (22R)- and (22S)-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione 16α,17α-acetals. Preparation of (22RS)-, (22R)- and (22S)-16α,17α-butylidenedioxy-6α,9α-difluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione. A . To a suspension of 1.0 g of 6α,9α-difluoro-11β,16α,17α,21-tetra-hydroxypregna-1-4-diene-3,20-dione in 500 ml of methylene chloride 0.32 ml of freshly distilled n -butanal and 2 ml of 72% perchloric acid were added. The reaction mixture was allowed to stand for 24 h at room temperature under stirring. The reaction mixture was washed with 10% aqueous potassium carbonate solution and water, dried over sodium sulphate and evaporated. The residue was dissolved in ethyl acetate and precipitated with petroleum ether leaving 883 mg of (22RS)-16α,17α-butylidenedioxy-6α,9α-difluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione. HPLC-analysis showed 99% purity and the ratio 16:84 between the 22S- and 22R-epimers. Molecular weight: 466 (calculated 466.5). The (22RS) epimeric mixture was chromatographed on Sephadex LH-20 column (76x6.3 cm) using heptane:chloroform:ethanol, 20:20:1, as mobile phase. The fractions 12315-13425 ml (A) and 13740-15690 ml (B) were collected and evaporated and the residue dissolved in methylene chloride and precipitated with petr.-ether. Fraction A gave 62 mg of (22S)- and fraction B 687 mg of (22R)-16α,17α-butylidenedioxy-6α,9α-difluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione. The (22S)-epimer: Molecular weight 466 (calculated 466.5), m.p. 196-200°C. The (22R)-epimer: Molecular weight 466 (calculated 466.5), m.p. 169-72°C. B. To a solution of 1.0 g of 6α,9α-difluoro-11β,21-dihydroxy-16α,17α-[(1-methylethylidene)bis(oxy)] pregna-1,4-diene-3,20-dione in 500 ml of methylene chloride was added 0.30 ml freshly distilled n-butanal and 2 ml of 72% perchloric acid. The reaction mixture was allowed to stand for 24 h at 33°C under stirring, extracted with aqueous potassium carbonate and water, dried over sodium sulphate and evaporated. The residue was dissolved in methylene chloride and precipitated with petr.-ether yielding 848 mg of (22RS)-16α,17α-butylidenedioxy-6α,9α-difluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione. HPLC-analysis showed 93% purity and the ratio 12/88 between the 22S- and 22R-epimers. B'. To a suspension of 4.0 g of 6α,9α-difluoro-11β,21-dihydroxy-16α,17α-[(methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione in 100 ml of heptane was added 1.2 ml of freshly distilled n-butanal and 3.8 ml of perchloric acid (72%]. The reaction mixture was allowed to stand for 5 h at room temperature under vigorous stirring, extracted with aqueous potassium carbonate and water, dried over sodium sulphate and evaporated yielding 4.0 g of (22RS)-16α,17α-butylidenedioxy-6α,9α-difluoro-11β,21-dihydroxypregna-1,4-diene-3,20-dione HPLC-analysis showed 98.5% purity and the ratio 3/97 between the 22S- and 22R-epimers. After two recrystallisations from chloroform-petroleum ether 3.1 g of 22R-epimer was obtained, which contained only 1.1% of the 22S-epimer and 1.3% of other impurities. C. Similarly, by following the procedure set forth in the example by substituting 6α,9α-difluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione for 11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione, 9α-fluoro- and 6α-fluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione or the corresponding 16α,17α-acetonides non-fluorinated and fluorinated non-symmetric (22RS)-, (22R)- and (22S)-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione 16α,17α-acetals from acetaldehyde, propanal, butanal, isobutanal, pentanal, 3-methylbutanal, 2,2-dimethylpropanal, hexanal, heptanal, octanal, nonanal and dodecanal are prepared. Example 2 A. Prednacinolon 16α,17α-acetonide (250 mg;0,6 mmol) was dissolved in 75 ml of CH₂Cl₂. n-Butanal (130 mg; 1,8 mmol and 70% perchloric acid (0,025ml) were added. The solution was stirred at 33°C for 15 hours. The yellow solution was washed with 2x10 ml of 10% K₂CO₃ and 4x10 ml of H₂O, dried and evaporated. Yield: 257 mg (97,7%). HPLC gave 91,1% purity. Unreacted acetonide consists of 7,4% of the impurities. Epimer ratio 14,6/85,4. B. Triamcinolon 16α,17α-acetonide (0,5 g; 1,1 mmol) was dissolved in 150 ml of CH₂Cl₂. n-Butanal (260 mg; 3,6 mmol) and 70% perchloric acid (0,22 ml) were added. The mixture way stirred at 33°C for 16 hours. CH₂Cl₂ was taken over into a separation funnel and the reaction flask was washed several times with 10 ml K₂CO₃ and CH₂Cl₂, respectively. The solution was then washed with 2x10 ml of 10% K₂CO₃ and 4x10 ml of H₂O, dried and evaporated. Yield: 438 mg (84,9%). HPLC gave 80,2% purity. Epimer ratio 19/81. C. Fluocinolon 16α,17α-acetonide (0,5 g; 1,1 mmol) was dissolved in 150 ml of CH₂Cl₂. n-Butanal (260 mg; 3,6 mmol) and 70% perchloric acid (0,22 ml)were added. The mixture was stirred at 33°C for 24 hours. The CH₂Cl₂ phase was taken over into a separation funnel. The reaction flask was washed several times with 15 ml of 10% K₂CO₃ and CH₂Cl₂, respectively The solution was washed with 2x15 ml of 10% K₂CO₃ and 4x15 ml of H₂O, dried and evaporated. Yield: 513 ng (100%). HPLC gave 97,4% purity. Epimeric ratio 8.6/91,4. Example 3. This example sets forth a process for preparing 11β-hydroxy-16α,17α- [(1-methylethylidene)bis(oxy)] - and (20RS)-, (20R)- and (20S)-11β-hydroxy-16α,17α-alkylmethylenedioxyandrosta-1,4-diene-3-one-17β-carboxylic and -4-ene-3-one-17β-carboxylic acids. Preparation of 6α,9α-difluoro-11β-hydroxy-16a,17α-[(1-methylethylidene)-bis(oxy)]androsta-1,4-diene-3-one-17β-carboxylic acid. A . To a solution of 1.99 g of fluocinolone 16α,17α-acetonide in 120 ml of methanol 40 ml of 20% aqueous potassium carbonate was added. A stream of air was bubbled through this solution for about 20 h under stirring at room temperature. The methanol was evaporated and 200 ml of water was added to the residue. The solution was extracted with methylene chloride. The aqueous phase was acidified with diluted hydrochloric acid. The precipitate formed was collected by filtration and dried to yield 1.34 g of 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)-bis(oxy)]androsta-1,4-diene-3-one-17β-carboxylic acid, melting point 264-68°C, molecular weight 438. The purity determined by HPLC was 94.0%. The aqueous phase was extracted with ethyl acetate. After drying the solvent was evaporated leaving another 0.26 g portion of acid. Purity: 93.7%. B . Periodic acid (15.1 g) in 16.5 ml of water was added to a solution of fluocinolone 16α,17α-acetonide (5.0 g) in 55 ml dioxane. The reaction mixture was stirred at room temperature for 20 h, neutralized with saturated aqueous sodium hydrogen carbonate and evaporated. The residue was dissolved in 200 ml of methylene chloride and washed with 8 x 100 ml 10% aqueous potassium carbonate. The aqueous phase was acidified with conc. hydrochloric acid and extracted with 6 x 100 ml of ethyl acetate. After drying the solvent was evaporated. The residue was dissolved in 400 ml of ethyl acetate and precipitated with petroleum ether yielding 3.96 g of 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis-(oxy)]androsta-1,4-diene-3-one-17β-carboxylic acid. The purity determined by HPLC was 99.5%. C . Similarly, by following the procedure set forth in the example by substituting fluocinolone 16α,17α-acetonide for 11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione, 6α-fluoro-11β,16α,17α,21-tetrahydroxypregna-1,4-diene-3,20-dione, and triamcinolone 16α,17α-acetonide 11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17β-carboxylic acids are prepared. By substituting the 16α,17α-acetonide group for 16α,17α-acetals between 16α-hydroxyprednisolone 6α-fluor-16α-hydroxyprednisolone, triamcinolone and fluocinolone and acetaldehyde, propanal, butanal, isobutanal, pentanal, 3-methylbutanal, 2,2-dimethylpropanal, hexanal, heptanal, octanal, nonanal and dodecanal and their 21-esters (20RS)- (20R)- and (20S)-11β-hydroxy-16α,17α-alkyl-methylenedioxyandrosta-1,4-diene- and 4-ene-3-one-17β-carboxylic acids are prepared. Example 4 . 1'-Ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methyl ethyl idene)bis(oxy)]androsta-1,4-diene-3-one-17β-carboxylate. A . 6α,9α-Difluou-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17β-carboxylic acid (600 mg) and potassium hydrogen carbonate (684 mg) were dissolved in 45 ml of dimethyl formamide. 1-Bromoethyl ethyl carbonate (2 ml) was added and the reaction mixture stirred at room temperature overnight. Water (200 ml) was added and the mixture was extracted with methylene chloride. The combined extracts were washed with 5% aqueous sodium hydrogen carbonate and water, and the residue purified by chromatography on a Sephadex LH-20 column (72x6.3 cm) using chloroform as mobil phase. The fraction 1515-2250 ml was collected and evaporated yielding 480 mg of 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17β-carboxylate. The purity determined by HPLC was 98.1% and the ratio epimer A/B, 48/52. Melting point: 218-27°C. [α] 25 / D = +63.2° (c=0.214; CH₂Cl₂). The molecular weight was 554. The 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17β-carboxylate (480 mg) was chromatographed on a Sephadex LH-20 column (76x6.3 cm) using heptane:chloroform:ethanol, 20:20:1, as mobile phase. The fraction 2325-2715 ml was collected, evaporated and the residue dissolved in methylene chloride and precipitated by petroleum ether giving 200 mg of a compound (A) of purity 97.3% (determined by HPLC analysis). Melting point: 246-50°C. [α] 25 / D = +100.5° (c=0.214; CH₂Cl₂). The molecular weight was 554. The fraction 4140-5100 ml yielded 250 mg of a compound (B) with purity 99.0%. Melting point: 250-55°C. [α] 25 / D = +28.5° (c=0.246; CH₂Cl₂). The molecular weight was 554. The methine signal from the ester group is shifted 0.13 ppm downfield in ¹H-NMR spectrum of B compared to A, while the rest of the spectra are nearly identical. The electron impact mass spectra of A and B are identical apart from the intensities of the mass peaks. These spectroscopic differences and similarities indicate that A and B are epimers due to the chiral centre in the ester group. B. 6α,9α-Difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid(200 mg) was dissolved in 25 ml of dimethylformamide. 1-Chloroethyl ethyl carbonate (100 mg), potassium hydrogen carbonate (70 mg) and 18-crown-6-ether were added. The reaction mixture was stirred at 80°C for 3 h, cooled, extracted with methylene chloride after addition of 150 ml of water, dried and evaporated. The crude product was purified in the same way as in procedure A leaving 207 mg of 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)] androsta-1,4-diene--3-one-17β-carboxylate. The purity (HPLC) was 98. 4% and the ratio epimer A/B, 54/46. C. 6α,9α-Difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid (200 mg) and 1,5-diazabicyclo [5.4.0] undecene-5 (140 mg) were suspended in 25 ml of benzene and warmed to reflux. A solution of 1-bromoethyl ethyl carbonate (175 mg) in 5 ml of benzene was added and the mixture was refluxed for 2 1/2 h. After cooling 50 ml of methylene chloride was added and the solution was washed with water, dried and evaporated. The crude product was purified in the same way as in procedure A, yielding 207 mg of 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α- [(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylate. The purity (HPLC) was 96.4% and the ratio epimer A/B, 44/56. D. To a solution of 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid (100 mg) in 25 ml of acetone 175 mg of α-bromodiethylcarbonate and 45 mg of anhydrous potassium carbonate were added. The mixture was heated for 6 h at reflux. The cooled reaction mixture was poured into 150 ml of water and extracted with methylene chloride. The extract was washed with water, dried over sodium sulphate and evaporated yielding 65 mg of solid 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α, [(1methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylate. The purity determined by HPLC was 97.6% and the ratio epimer A/B, 49/51. E. 6α,9α-Difluoro-11β-hydroxy-16α,17α- [(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid (500 mg) and tetrabutylammonium hydrogen sulphate (577 mg) were added to 3 ml of 1M sodium hydroxide.A solution of 435 mg of 1-bromoethyl ethyl carbonate in 50 ml of methylene chloride was added. The mixture was refluxed with stirring overnight. The two layers were separated. The organic layer was washed with 2x10 ml of water, dried and evaporated. The crude product was purified by chromatography on a Sephadex LH-20 column (72x6.3 cm) using chloroform as mobile phase. The fraction 1545-1950 ml was collected and evaporated and the residue precipitated from methylene chloride - petroleum ether leaving 341 mg of 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)-bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylate. The purity determined with HPLC was 99.2% and the ratio epimer A,B, 56/44. F. 6α,9α-Difluoro-11β-hydroxy-16α,17α- [(1-methylethylidene)bis-(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid (200 mg) and tricaprylmethylammonium chloride (200 mg) were added to 5 ml of saturated aqueous NaHCO₃. A solution of 100 mg of 1-bromoethyl ethyl carbonate in 10 ml of methylene chloride was added. The mixture was stirred at 45°C for 20 h, diluted with 10 ml of methylene chloride and isolated and purified in the same way as in procedure E yielding 254 mg of 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy--16α,17α- [(1-methylethylidene)bis(oxy)] - androsta-1,4-diene-3-one-17β-carboxylate. The purity (HPLC) was 97.4% and the ratio epimer A/B, 60/40. G. 6α,9α-Difluoro-11β-hydroxy-16α,17α- [(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid (200 mg), 1-bromoethyl ethyl carbonate (135 mg) and triethylamine (275 mg) were dissolved in 20 ml of dimethylformamide. The mixture was stirred at 80°C for 3 h, diluted with 200 ml of methylene chloride, washed with water, dried and evaporated. The crude product was purified in the same way as in procedure A yielding 69 mg of 1'-ethoxycarbonyloxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α- [(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylate. The purity (HPLC) was 97.8% and the ratio epimer A/B, 48/52. Exempel 5. 1'-Acetoxyethyl 6α,9α-difluoro-11β-hydroxy-16α,17α-[(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylate. 6α,9α-Difluoro-11β-hydroxy-16α,17α- [(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17β-carboxylic acid (500 mg) and potassium hydrogen carbonate (575 mg) were dissolved in 40 ml of dimethylformamide. 1-chloroethyl acetate (1 ml) was added and the reaction mixture was stirred at room temperature for 40 h. The reaction mixture was poured into 50 ml of water and extracted with methylene chloride. The extract was washed with aqueous sodium hydrogen carbonate and water, dried and evaporated. The residue was chromatographed on Sephadex LH-20 column (72x6.3 cm) using chloroform as mobile phase. The fractions 1755-2025 and 2026-2325 ml were collected and evaporated. The solid product from fraction 1755-2025 ml was further purified by chromatography on a sephadex LH-20 column (76x6.3 cm i.d.) using a mixture of heptane-chloroform-ethanol, 20:20:1, as mobile phase. The fraction 2505-2880 ml was collected and evaporated. the residue was dissolved in methylene chloride and precipitated with petroleum ether leaving 167 mg of solid product (A). The purity determind by HPLC was 99.1%. Melting point 238-59°C. [α] 25 / D = +94° (c=0.192; CH₂Cl₂). The molecular weight was 524. The solid product from fraction 2026-2325 ml above was further purified by chromatography in the same way as above. The fraction 5100-5670 ml was collected and evaporated. The residue was dissolved in methylene chloride and precipitated with petroleum ether yielding 165 mg of solid product (B). The purity determined with HPLC was 99.4%. Melting point 261-65°C. [α] 25 / D = +34° (c=0.262; CH₂Cl₂). The molecular weight was 524. The ¹H-NMR spectra of A and B are nearly identical with the exception of the methine quartet from the ester group which is shifted 0.16 ppm downfield in compound B compared to A. The fragmentation patterns of A and B in electron impact mass spectra are identical apart from the intensities of the mass peaks. These spectroscopic properties of A and B indicate that they are epimers due to the chiral centre in the ester group. Exemple 6-88. The substance given in Table 1-3 below were prepared, isolated and purified in a manner analogous to that described in Examples 4 and 5. Example 89. Pharmaceutical Preparations The following non-limitative examples illustrate formulations intended for different topical forms of administration. The amount of active steroid in the percutaneous formulations are ordinarily 0.001-0.2% (w/w), preferably 0.01-0.1% (w/w). Formulation 1, Ointment Steroid, micronized 0.025 g Liquid paraffin 10.0 g White soft paraffin ad 100.0 g Formulation 2, Ointment Steroid 0.025 g Propylene glycol 5.0 g Sorbitan sesquioleate 5.0 g Liquid paraffin 10.0 g White soft paraffin ad 100.0 g Formulation 3, Oil in water cream Steroid 0.025 g Cetanol 5.0 g Glyceryl monostearate 5.0 g Liquid paraffin 10.0 g Cetomacrogol 1000 2.0 g Citric acid 0.1 g Sodium citrate 0.2 g Propylene glycol 35.0 g Water ad 100.0 g Formulation 4, Oil in water cream Steroid, micronized 0.025 g White soft paraffin 15.0 g Liquid paraffin 5.0 g Cetanol 5.0 g Sorbimacrogol stearate 2.0 g Sorbitan monostearate 0.5 g Sorbic acid 0.2 g Citric acid 0.1 g Sodium citrate 0.2 g Water ad 100.0 g Formulation 5, Water in oil cream Steroid 0.025 g White soft paraffin 35.0 g Liquid paraffin 5.0 g Sorbitan sesquioleate 5.0 g Sorbic acid 0.2 g Citric acid 0.1 g Sodium citrate 0.2 g Water ad 100.0 g Formulation 6, Lotion Steroid 0.25 mg Isopropanol 0.5 ml Carboxyvinylpolymer 3 mg NaOH q.s. Water ad 1.0 g Formulation 7, Suspension for injection Steroid, micronized 0.05-10 mg Sodium carboxymethylcellulose 7 mg NaCl 7 mg Polyoxyethylene (20) sorbitan monoleate 0.5 mg Phenyl carbinol 8 mg Water, sterile ad 1.0 ml Formulation 8, Aerosol for oral and nasal inhalation Steroid, micronized 0.1 % w/w Sorbitan trioleate 0.7 % w/w Trichlorofluoromethane 24.8 % w/w Dichlorotetrafluoromethane 24.8 % w/w Dichlorodifluoromethane 49.6 % w/w Formulation 9, Solution for atomization Steroid 7.0 mg Propylene glycol 5.0 g Water ad 10.0 g Formulation 10, Powder for inhalation A gelatin capsule is filled with a mixture of Steroid, micronized 0.1 mg Lactose 20 mg The powder is inhaled by means of an inhalation device. Pharmacology The affinity of the mew androstane-17β-carboxylic acid esters to the glucocorticoid receptor All steroids according to formula I are physiologically active compounds. The affinity of the novel androstane-17β-carboxylic acid esters to the glucocorticoid receptor has been used as a model for determination of the anti-inflammatory potency. Their receptor affinities have been compared to budesonide ([22R,S]-16α,17α-butylidenedioxy-11β,21-dihydroxypregna-1,4-diene-3,20-dione) a highly active glucocorticoid with a favourable ratio between local and systemic effects (Thalén and Brattsand, Arzneim.-Forsch. 29 , 1687-90 (1979)). Male Sprague-Dawley rats, one to two months of age, were used throughout the investigation. The thymus was removed and put into ice-cold saline. The tissue was homogenized in a Potter Elvehjem homogenizer in 10 ml of a buffer containing 20 mM Tris, pH 7.4, 10% (w/v) glycerol, 1 mM EDTA, 20 mM NaMoO₄, 10 mM mercaptoethanol. The homogenate was centrifuged for 15 min at 20,000 x g. Portions of the 20,000 x g supernatant (230 µl) were incubated for about 24 h at 0°C with 100 µl phenylmethylsulphonylfluoride (an esterase inhibitor, final conc. 0.5 mM), 20 µl unlabelled competitor and 50 µl ³H-labelled dexamethasone (final conc. 3 nM). Bound and free steroid were separated by incubating the mixture with 60 µl 2.5% (w/v) charcoal and 0.25% (w/v) dextran T70 suspension in 20 mM Tris, pH 7.4, 1 mM EDTA, and 20 mM NaMoO₄ for 10 min at 0°C. Following a centrifugation at 500 x g for 10 min, 230 µl of the supernatant was counted in 10 ml Insta-Gel in a Packard scintillation spectrophotometer. The supernatants were incubated with a) [³H]dexamethasone alone, b) [³H]dexamethasone plus 1000 fold excess of unlabelled dexamethasone and c) [³H]dexamethasone plus 0.03-300 fold "excess" of competitor. The nonspecific binding was determined when 1000 fold excess of unlabelled dexamethasone was added to [³H]-labelled dexamethasone. The radioactivity bound to the receptor in the presence of competitor divided by the radioactivity bound to the receptor in the absence of competitor multiplied by 100 gives the percentage specific binding of labelled dexamethasone. For each concentration of a competitor the percentage specifically bound radioactivity is plotted against the log of concentration of competitor. The curves are compared at the 50% specific binding level and referenced to budesonide, which is assigned a relative binding affinity (RBA) of 1. Table summarizing relative binding affinities (RBA) to the glucocorticoid receptor of some of the investigated componds. Compound according to Ex. No. RBA Budesonide 1 4 epimer B 0.30 5 epimer B 0.17 27 0.50 38 0.04 55 0.20 64 0.05 67 0.04 69 0.44 84 1.03 87 0.63

Claims: A process for the preparation of a compound of the formula or a stereoisomeric compound thereof, in which formula the 1,2 position is saturated or is a double bond X₁ is selected from hydrogen, fluorine, chlorine and bromine X₂ is selected from hydrogen, fluorine, chlorine and bromine R₂ is selected from straight and branched hydrocarbon chains having 1-10 carbon atoms and R₇ is hydrogen or an acyl group with 1-10 carbon atoms arranged in a straight or branched chain, characterized by reaction of a compound of the formula with a compound of the formula wherein X₁, X₂, R₂, R₇ and have the meaning given above, in the presence of perchloric acid. A process according to claim 1 wherein a compound of formula D , wherein X₁ is hydrogen, X₂ is hydrogen, R₂ is a straight hydrocarbon chain having 3 carbon atoms, R₇ is hydrogen and the 1,2-position is a double bond, is prepared.

Inventors: ANDERSSON PAUL HAKAN, ANDERSSON PER TURE, AXELSSON BENGT INGEMAR, THALEN BROR ARNE, TROFAST JAN WILLIAM, ANDERSSON, PAUL HAKAN, ANDERSSON, PER TURE, AXELSSON, BENGT INGEMAR, THALEN, BROR ARNE, TROFAST, JAN WILLIAM
Applicants: DRACO AB, AKTIEBOLAGET DRACO
Application year: 1986
Main classification: C07J3
IPC codes: C07J71