16,17-acetalsubstituted androstane-17.beta.-carboxylic acid esters prossessing high binding affinity to the glucocorticosteroid receptor

DESCRIPTION
1. Field of the Invention
The present invention relates to novel, pharmacologically active compounds and to intermediates and a process for their use in mammals, including humans. The invention also relates to pharmaceutical compositions containing the compounds and to methods of treatment of inflammatory, allergic, muscoskeletal or dermatological conditions with these compounds.
The object of the invention is to provide a glucocorticosteroid which possesses high anti-inflammatory potency on the place of application and low glucocorticoid systemic potency.
2. Background Art
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, expecially 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, 16.alpha.,17.alpha.-acetalsubstituted glucocorticoids. E. Dahlberg, A. Thalen, R. Brattsand, J-A Gustafsson, U. Johansson, K. Roempke, and T. Saartok, Mol. Pharmacol. 25 (1984), 70.]
DISCLOSURE OF THE INVENTION
The present invention is based on the observation that certain 3-oxo-androsta-1,4-diene-17.beta.-carboxylic acid esters possess high binding affinity to the glucocorticosteroid receptor. The compounds of the invention can be used for the treatment and control of inflammatory conditions.
The compounds of the invention are characterized by the formula ##STR4## wherein the 1,2-position is saturated or is a double bond
X.sub.1 is selected from hydrogen, fluorine, chlorine and bromine
X.sub.2 is selected from hydrogen, fluorine, chlorine and bromine
R.sub.1 is selected from hydrogen or a straight or branched hydrocarbon chain having 1-4 carbon atoms
R.sub.2 is selected from hydrogen or straight and branched hydrocarbon chains having 1-10 carbon atoms and
R.sub.3 is selected from ##STR5## Y is O or S R.sub.4 is selected from hydrogen, straight or branched hydrocarbon chains having 1-10 carbon atoms or from phenyl
R.sub.5 is selected from hydrogen or methyl and
R.sub.6 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, --(CH.sub.2).sub.m CH(CH.sub.2).sub.n (m=0,1,2; n=2,3,4,5,6), 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 R.sub.2 is hydrogen R.sub.1 is methyl.
The individual stereoisomeric components present in a mixture of a steroid having the above formula (I) can be elucidated in the following way: ##STR6##
The individual stereoisomeric components present, in a mixture of steroid 17.beta.-carboxylic acid esters having the formulas ##STR7## where St is the steroid moiety, can be elucidated in the following way ##STR8##
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 above 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.
Particular compounds of the invention which are preferred:
1'-Ethoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17.beta.-carboxylate, the epimeric mixture A+B and epimer B.
1'-isopropoxycarbonyloxyethyl 9.alpha.-fluoro-11.beta.-hydroxy-15.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17.beta.-carboxylate, epimer B.
1'-proposycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androxta-1,4-diene-3-one-17.alpha.-carboxylate, epimer B.
1'-isopropoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate, epimeric mixture A+B and epimer B.
1'-Acetoxyethyl (20R)-9.alpha.-fluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-propylmethylenedioxyandrosta-1,4-diene-3-one-17.beta.-carboxylate, epimer B.
1'-Ethoxycarbonyloxyethyl (22R)-9.alpha.-fluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-propylmethylenedioxyandrosta-1,4-diene-3-one-17.beta.-carboxylate, epimer B.
1'-isopropoxycarbonyloxyethyl (20R)-9.alpha.-fluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-propylmethylenedioxyandrosta-1,4-diene-3-one-17.beta.-carboxylate, epimer B.
1'-Ethoxycarbonyloxyethyl (20R)-6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-propylmethylenedioxyandrosta-1,4-diene-3-one-17.beta.-carboxylate, epimeric mixture A+B and epimer B.
METHODS OF PREPARATION
The compounds of the invention are prepared by the oxidation of a compound of the formulas X, XI and XII to the corresponding 17.beta.-carboxylic acid: ##STR9## wherein the solid and broken lines between C-1 and C-2 represent a single or double bond, X.sub.1, X.sub.2, R.sub.1 and R.sub.2 have the meaning given above, and R.sub.7 is hydrogen or an acyl group with 1-10 carbon atoms arranged in a straight or branched chain,
The 17.beta.-carboxylic acids then are esterified to give compounds characterized by the formula I-IX, wherein X.sub.1, X.sub.2, R.sub.1, R.sub.2 and R.sub.3 have the meaning given above.
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 ethenol 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 preferred. The conversion of a compound of formula X, XI or XII to a 17.beta.-carboxylic acid of formula I, II, or III (R.sub.3 =H) takes place at ambient temperatures, i.e. 20.degree.-25.degree. 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 17.beta. side-chain of compounds of formula X, XI and XII to the corresponding 17.beta. 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 17.beta.-carboxylic acids of compounds of formula I, II and III (R.sub.3 =H) may be esterified in known manner to provide 17.beta. carboxylate esters according to the invention. For example, the 17.beta.-carboxylic acid may be reacted with an appropriate alcohol and a carbodiimide, e.g. dicylohexylcarbodiimide, in a suitable solvent such as diethylether, tetrahydrofurane, methylene chloride or pyridine advantageously at a temperature of 25.degree.-100.degree. C. Alternatively, a salt of the 17.beta.-carboxylic acid with an alkali metal, e.g. lithium, sodium or potassium, a salt of a quaternary ammonium compound, such as a salt or 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, methylethylketone, or dimethyl formamide, dimethyl sulphoxide, mthylenechloride or chloroform, conveniently at a temperature in the range 25.degree.-100.degree. C. The reaction may also be performed in the presence of a crown ether.
The crude steroid ester derivatives formed are after isolation purified by chromatography on a suitable material, for instance cross-linked dextran gels of Sephadex.RTM. 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 of the 16.alpha.,17.alpha.-hydroxy groups or at the esterification of the 17.beta.-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 and V above are subjected to column chromatography, 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.RTM. LH, e.g. Sephadex.RTM. LH-20 in combination with a suitable organic solvent as eluting agent. Sephadex.RTM. LH-20, prepared by Pharmacia Fine Chemicals AB, Uppsala, Sweden, is a beadformed hydroxyproylated 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.
As starting materials for the compounds of the invention compounds of the formulas X, XI and XII are used. They are prepared by reaction of compounds with the formula ##STR10## wherein the solid and broken lines between C-1 and C-2 represent a single or double bond, and X.sub.1, X.sub.2 and R.sub.7 have the meaning given above, with an aldehyde of the formula ##STR11## wherein R.sub.2 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 an acid catalyst, e.g. perchloric acid, p-toluene sulphonic acid, hydrochloric acid in an ether, preferably dioxane, or halogenated hydrocarbons, preferably methylene chloride or chloroform.
Compounds X, XI and XII are also prepared by transacetalisation of the corresponding 16.alpha.,17.alpha.-acetonides ##STR12## wherein the solid and broken lines between C.sub.1 and C.sub.2 represent a single or double bond and X.sub.1, X.sub.2 and R.sub.7 have the meaning given above with an aldehyde of the formula ##STR13## wherein R.sub.2 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 a strong inorganic acid as catalyst, preferably perchloric or hydrochloric acid, 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 the invention 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 continuous 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 .mu.g, preferably 20-250 .mu.g 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 .mu.m, which are suspended in a propellent mixture with the assistance of a dispersant, such as sorbitan trioleate, oleic acid, lecithin or sodium salt or 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/cm.sup.2.h.sup.-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 .mu.Bondapak C.sub.18 column (300.times.3.9 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 (b 22S)-11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione 16.alpha.,17.alpha.-acetals.
Preparation of (22RS)-, (22R)- and (22S)-16.alpha.,17.alpha.-butylidenedioxy-6.alpha.,9.alpha.-difluoro-11.beta.,21-dihydroxypregna-1,4-diene-3,20-dione.
A. To a suspension of 1.0 g of 6.alpha.,9.alpha.-difluoro-11.beta.,16.alpha.,17.alpha.,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)-16.alpha.,17.alpha.-butylidenedioxy-6.alpha.,9.alpha.-difluoro-11.beta.,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 (76.times.6.3 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)-16.alpha.,17.alpha.-butylidenedioxy-6.alpha.,9.alpha.-difluoro-11.alpha.,21-dihydroxypregna-1,4-diene-3,20-dione. The (22S)-epimer: Molecular weight 466 (calculated 466.5), m.p. 196.gtoreq.-200.degree. C. The (22R)-epimer: Molecular weight 466 (calculated 466.5), m.p. 169.degree.-72.degree. C.
B. To a solution of 1.0 g of 6.alpha.,9.alpha.-difluoro-11.beta.,21-dihydroxy-16.alpha.,17.alpha.-[(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)-16.alpha.,17.alpha.-butylidenedioxy-6.alpha.,9.alpha.-difluoro-11.beta.,21-dihydroxypregna-1,4-diene-3,20-dione. HPLC-analysis showed purity and the ratio 12/88 between the 22S- and 22R-epimers.
B'. To a suspension of 4.0 g of 6.alpha.,9.alpha.-difluoro-11.beta.,21-dihydroxy-16.alpha.,17.alpha.-[(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)-16.alpha.,17.alpha.-butylidenedioxy-6.alpha.,9.alpha.-difluoro-11.beta.,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 recrystalisations 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 6.alpha.,9.alpha.-difluoro-11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione for 11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione, 9.alpha.-fluoro- and 6.alpha.-fluoro-11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione or the corresponding 16.alpha.,17.alpha.-acetonides non-fluorinated and fluorinated non-symmetric (22RS)-, (22R)- and (22S)-11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione 16.alpha.,17.alpha.-acetals from acetaldehyde, propanal, butanal, isobutanal, pentanal, 3-methylbutanal, 2,2-dimethylpropanal, hexanal, heptanal, octanal, nonanal, and dodecanal are prepared.
EXAMPLE 2
A. Prednacinolon 16.alpha.,17.alpha.-acetonide (250 mg; 0,5 mmol) was dissolved in 75 ml of CH.sub.2 Cl.sub.2. n-Butanal (130 mg; 1,8 mmol and 70% perchloric acid (0,025 ml) were added. The solution was stirred at 33.degree. C. for 15 hours. The yellow solution was washed with 2.times.10 ml of 10% K.sub.2 CO.sub.3 and 4.times.10 ml of H.sub.2 O, 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. Trimcinolon 16.alpha.,17.alpha.-acetonide (0,5 g; 1,1 mmol) was dissolved in 150 ml of CH.sub.2 Cl.sub.2. n-Butanal (260 mg; 3,6 mmol) and 70% perchloric acid (0,22 ml) were added. The mixture way stirred at 33.degree. C. for 16 hours. CH.sub.2 Cl.sub.2 was taken over into a separation funnel and the reaction flask was washed several times with 10 ml K.sub.2 CO.sub.3 and CH.sub.2 Cl.sub.2, respectively. The solution was then washed with 2.times.10 ml of 10% K.sub.2 CO.sub.3 and 4.times.10 ml of H.sub.2 O, dried and evaporated. Yield: 438 mg (84,9%). HPLC gave 80,2% purity. Epimer ratio 19/81.
C. Fluocinolon 16.alpha.,17.alpha.-acetonide (0,5 g; 1,1 mmol) was dissolved in 150 ml of CH.sub.2 Cl.sub.2. n-Butanal (260 mg; 3,6mmol) and 70% perchloric acid (0,22 ml) were added. The mixture was stirred at 33.degree. C. for 24 hours. The CH.sub.2 Cl.sub.2 phase was taken over into a separation funnel. The reacton flask was washed several times with 15 ml of 10% K.sub.2 CO.sub.3 and CH.sub.2 Cl.sub.2, respectively. The solution was washed with 2.times.15 ml of 10% K.sub.2 CO.sub.3 and 4.times.15 ml of H.sub.2 O, dried and evaporated. Yield: 513 mg (100%). HPLC gave 97,4% purity. Epimeric ratio 8.6/91,4.
EXAMPLE 3
This example sets forth a process for preparing 11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]- and (20RS)-, (20R)- and (20S)-11.beta.-hydroxy-16.alpha.,17.alpha.-alkylmethylenedioxyandrosta-1,4-diene-3-one-17.beta.-carboxylic and -4-ene-3-one-17.beta.-carboxylic acids.
Preparation of 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)-bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylic acid.
A. To a solution of 1.99 g of fluocinolone 16.alpha.,17.alpha.-acetonide in 120 ml of methanol 40 ml of 20% aqueous potassium carbonate was added. A stream of air was bubbled though 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 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)-bis(oxy)androsta-1,4-diene-3-one-17.beta.-carboxylic acid, melting point 264.degree.-68.degree. 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 16.alpha.,17.alpha.-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.times.100 ml 10% aqueous potassium carbonate. The aqueous phase was acidified with conc. hydrochloric acid and extracted with 6.times.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 3.69 g of 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)androsta-1,4-diene-3-one-17.beta.-carboxylic acid. The purity determined by HPLC was 99.5%.
C. Similarly, by following the procedure set forth in the example by substituting fluocinolone 16.alpha.,17.alpha.-acetonide for 11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione, 6.alpha.-fluoro-11.beta.,16.alpha.,17.alpha.,21-tetrahydroxypregna-1,4-diene-3,20-dione, and triamcinolone 16.alpha.,17.alpha.-acetonide 11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylic acids are prepared. By substituting the 16.alpha.,17.alpha.-acetonide group for 16.alpha.,17.alpha.-acetals between 16.alpha.-hydroxyprednisolone 6.alpha.-fluor-16.alpha.-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 (20R)-11.beta.-hydroxy-16.alpha.,17.alpha.-alkylmethylenedioxyandrosta-1,4-diene- and 4-ene-3-one-17.beta.-carboxylic acids are prepared.
EXAMPLE 4
1'-Ethoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate.
A. 6.alpha.,9.alpha.-Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethlidene)bis(oxy)]-androsta-1,4-diene-3-one-17.beta.-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 (72.times.6.3 cm) using chloroform as mobil phase. The fraction 1515-2250 ml was collected and evaporated yielding 480 mg of 1'-ethoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity determined by HPLC was 98.1% and the ratio epimer A/B, 48/52. Melting point: 218.degree.-27.degree. C. [.alpha.].sub.D.sup.25 =+63.2.degree. (c=0.214; CH.sub.2 Cl.sub.2). The molecular weight was 554.
The 1'-ethoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-[11.beta.-hydroxy-16.alpha.,17.alpha.-(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate (480 mg) was chromatographed on a Sephadex LH-20 column (76.times.6.3 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.degree.-50.degree. C. [.alpha.].sub.D.sup.25 =+100.5.degree. (c=0.214; CH.sub.2 Cl.sub.2). The molecular weight was 554.
The fraction 4140-5100 ml yielded 250 mg of a compound (B) with purity 99.0%. Melting point: 250.degree.-55.degree. C. [.alpha.].sub.D.sup.25 =+28.5.degree. (c=0.246; CH.sub.2 Cl.sub.2). The molecular weight was 554. The methine signal from the ester group is shifted 0.13 ppm downfield in .sup.1 H-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 center in the ester group.
B. 6.alpha.,9.alpha.-Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis)oxy)]androsta-1,4-diene-3-one-17.beta.-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.degree. C. for 3 g, cooled, extracted with methylene chloride after addition of 150 ml of water, dried ad evaporated. The crude product was purified in the same way as in procedure A leaving 207 mg of 1'-ethoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.- [(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity (HPLC) was 98.4% and the ratio epimer A/B, 54/46.
C. 6.alpha.,9.alpha.-Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-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 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity (HPLC) was 96.4% and the ratio epimer A/B, 44/56.
D. To a solution of 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylic acid (100 mg) in 25 ml of acetone 175 mg of .alpha.-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 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha., [(1-methyolethylidene)bis(oxy)] androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity determined by HPLC was 97.6% and the ration epimer A/B, 49/51.
E. 6.alpha.,9.alpha.Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)] androsta-1,4-diene-3-one-17.beta.-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-bromethyl 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 2.times.10 ml of water, dried and evaporated. The crude product was purified by chromatography on a Sephadex LH-20 column (72.times.6.3 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 gm of 1'-ethoxycarbonyloxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.- [(1-methylethylidene)-bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity determined with HPLC was 99.2% and the ratio epimer A/B, 56/44.
F. 6.alpha.,9.alpha.-Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis-(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylic acid (200 mg) and tricaprylmethylammonium chloride (200 mg) were added to 5 ml of saturated aqueous NaHCO.sub.3. A solution of 100 mg of 1-bromoethyl ethyl carbonate in 10 ml of methylene chloride was added. The mixture was stirred at 45.degree. 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 6 .alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]-androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity (HPLC) was 97.4% and the ratio epimer A/B, 60/40.
G. 6.alpha.,9.alpha.-Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-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.degree. 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 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate. The purity (HPLC) was 97.8% and the ratio epimer A/B, 48/52.
EXAMPLE 5
1'-Acetoxyethyl 6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-one-17.beta.-carboxylate.
6.alpha.,9.alpha.-Difluoro-11.beta.-hydroxy-16.alpha.,17.alpha.-[(1-methylethylidene)bis(oxy)]androsta-1,4-diene-3-diene-3-one-17.beta.-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 (72.times.6.3 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 (76.times.6.3 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.degree.-59.degree. C. [.alpha.].sub.D.sup.25 =+94.degree. (C=0.192; CH.sub.2 Cl.sub.2). 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.degree.-65.degree. C. [.alpha.].sub.D.sup.25 =+34.degree. (c=0.262; CH.sub.2 Cl.sub.2). The molecular weight was 524.
The .sup.1 H-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 A and B indicate that they are epimers due to the chiral center in the ester group.
EXAMPLE 6.about.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.
TABLE 1__________________________________________________________________________ ##STR14## [.alpha.].sub.D.sup.25Example (c = 0.2 in Molecular Retentionno. X.sub.1 X.sub.2 R.sub.4 R.sub.5 R.sub.6 /YR.sub.6 Epimer Mp .degree.C. CH.sub.2 Cl.sub.2) calc. found volume__________________________________________________________________________ (ml) 6 H H fenyl H CH.sub.3 A 242 (dec) +79.degree. 550.7 550 1665-1890.sup.1 7 H H fenyl H CH.sub.3 B 221 (dec) +89.degree. 550.7 550 1891-2175.sup.1 8 F H CH(CH.sub.3).sub.2 H CH.sub.3 A -- +102.degree. 534.6 534 2325-2580.sup.1 9 F H CH(CH.sub.3).sub.2 H CH.sub.3 B -- +40.degree. 534.6 534 3165-3555.sup.110 F H fenyl H CH.sub.3 A 249 (dec) +73.degree. 568.6 568 2040-2355.sup.111 F H fenyl H CH.sub.3 B 238 (dec) +75.degree. 568.6 568 2895-3285.sup.112 F F CH.sub.3 H C(CH.sub.3).sub.3 A 262-70 +87.degree. 566.6 566 2190-2505.sup.113 F F CH.sub.3 H C(CH.sub.3).sub.3 B 268-77 + 50.degree. 566.6 566 3525-3990.sup.114 F F CH.sub.3 H fenyl A 224-30 +96.degree. 586.6 586 2325-2625.sup.115 F F CH.sub.3 H fenyl B 259-67 +48.degree. 586.6 586 4350-4875.sup.116 F F CH.sub.3 CH.sub.3 CH.sub.3 -- 130-42 +61.degree. 538.6 538 1965-2220.sup.117 F H CH.sub.3 H OC(CH.sub.3).sub.3 A 184-87 +98.degree. 564.7 564 235-280.sup.318 F H CH.sub.3 H OC(CH.sub.3).sub.3 B >300 +30.degree. 564.7 564 525-630.sup.219 H F CH.sub.3 H OCH(CH.sub.3).sub.2 A 250-53 +109.degree. 550.6 550 1530-1770.sup.120 H F CH.sub.3 H OCH(CH.sub.3).sub.2 B 230-35 +58.degree. 550.6 550 2295-2850.sup.121 F F CH.sub.3 H OCH.sub.3 A 235-42 +102.degree. 540.6 540 590-690.sup.222 F F CH.sub.3 H OCH.sub.3 B 225-33 +31.degree. 540.6 540 395-430.sup.323 F F CH.sub.3 H O(CH.sub.2).sub.2 CH.sub.3 A 224- 31 +106.degree. 568.6 568 410-495.sup.224 F F CH.sub.3 H O(CH.sub.2).sub.2 CH.sub.3 B 227-30 +28.degree. 568.6 568 690-900.sup.225 F F CH.sub.3 H OCH(CH.sub.3).sub.2 A + B 205-28 +59.degree. 568.6 568 1365-1560.sup.526 F F CH.sub.3 H OCH(CH.sub.3).sub.2 A 210-25 +95.degree. 568.6 568 400-475.sup.227 F F CH.sub.3 H OCH(CH.sub.3).sub.2 B 242-47 +31.degree. 568.6 568 625-780.sup.228 F F CH.sub.3 H OCH(CH.sub.2 CH.sub.3).sub.2 A 226-28 +95.degree. 596.7 596 1785-2085.sup.129 F F CH.sub.3 H OCH(CH.sub.2 CH.sub.3).sub.2 B 183-97 +30.degree. 596.7 596 3150-3600.sup.130 F F CH.sub.3 H OCH.sub.2 CH(CH.sub.2 CH.sub.3).sub.2 A 217-21 +89.degree. 610.7 610 1725-1980.sup.131 F F CH.sub.3 H OCH.sub.2 CH(CH.sub.2 CH.sub.3).sub.2 B 207-10 +30.degree. 610.7 610 3120-3480.sup.132 F F CH.sub.3 H OC(CH.sub.3).sub.3 A + B 170-78 +65.degree. 582.6 582 1290-1920.sup.533 F F CH.sub.3 H OC(CH.sub.3).sub.3 A 177-79 +100.degree. 582.6 582 255-310.sup.334 F F CH.sub.3 H OC(CH.sub.3).sub.3 B 190-92 +27.degree. 582.6 582 650-800.sup.235 F F CH.sub.3 H OCH.sub.2 C(CH.sub.3).sub.3 A + B 208-36 +60.degree. 596.7 596 1605-1995.sup.136 F F CH.sub.3 H OCH.sub.2 C(CH.sub.3).sub.3 A 248-56 +98.degree. 596.7 596 1845-2130.sup.137 F F CH.sub.3 H OCH.sub.2 C(CH.sub.3).sub.3 B 226-28 +28.degree. 596.7 596 3270-3750.sup.138 F F CH.sub.3 CH.sub.3 OCH.sub.2 CH.sub.3 -- -- -- 568.6 568 405-460.sup.2__________________________________________________________________________ .sup.1 On a Sephadex LH20 column (76 .times. 6.3 cm) using chloroformheptane-ethanol (20:20:1) as mobile phase. .sup.2 On a Sephadex LH20 column (87.5 .times. 2.5 cm) using chloroformheptane-ethanol (20:20:1) as mobile phase. .sup.3 On a Sephadex LH20 column (85 .times. 2.5 cm) using chloroform as mobile phase. .sup.4 On a Sephadex LH20 column (72 .times. 6.3 cm) using chloroform as mobile phase. .sup.5 On a Sephadex LH20 column (71.5 .times. 6.3 cm) using chloroform a mobile phase.
TABLE 2__________________________________________________________________________ ##STR15## [.alpha.].sub.D.sup.25Example (c = 0.2 in Molecular weight Retentionno. X.sub.1 X.sub.2 R.sub.2 R.sub.4 R.sub.6 /YR.sub.6 Epimer Mp .degree.C. CH.sub.2 Cl.sub.2) calc. found volume__________________________________________________________________________ (ml)39 H H CH.sub.3 H C(CH.sub.3).sub.3 -- 189-92 +78.degree. 502.6 502 1290-1665.sup.140 H H (CH.sub.2).sub.2 CH.sub.3 H CH.sub.3 -- 63-70 +79.degree. 488.6 488 1110-1260.sup.141 H H (CH.sub.2).sub.2 CH.sub.3 H C(CH.sub.3).sub.3 -- 192-96 +74.degree. 530.7 530 1245-1440.sup.142 F H (CH.sub.2).sub.2 CH.sub.3 H C(CH.sub.3).sub.3 -- 254-58 +64.degree. 548.7 548 1485-1800.sup.143 H H (CH.sub.2).sub.2 CH.sub.3 H O(CH.sub.2).sub.3 CH.sub.3 -- 40-46 +70.degree. 546.7 546 1200-1395.sup.144 H H (CH.sub.2).sub.2 CH.sub.3 H OC(CH.sub.3 ).sub.3 -- 155-58 +67.degree. 546.7 546 320-400.sup.245 H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 OCH.sub.2 CH.sub.3 A + B 163-75 +63.degree. 532.6 532 225-285.sup.246 F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 OCH.sub.2 CH.sub.3 A + B 138-60 -- 550.6 550 1410-1545.sup.147 F F (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 OCH.sub.2 CH.sub.3 A + B 160-87 -- 568.6 568 1620-2175.sup.1__________________________________________________________________________ .sup.1 On a Sephadex LH20 column (72 .times. 6.3 cm) using chloroform as mobile phase. .sup.2 On a Sephadex LH20 column (83 .times. 2.5 cm) using chloroform as mobile phase.
TABLE 3 ##STR16## [.alpha.] .sub.D.sup.25 Example (c = 0.2 in Molecular weight Retention no. X.sub.1 X.sub.2 R.sub.1 R.sub.2 R.sub.4 R.sub.5 R.sub.6 /YR.sub.6 Epimer Mp .degree.C. CH.sub.2 Cl.sub.2) calc. found volume (ml) 48H HCH.sub.3 HH HC(CH.sub.3).sub.3 -- 192-97 +67.degree. 502.6502 1 650-1995.sup.1 49 H H H CH.sub.3 H H C(CH.sub.3).sub.3 -- 196-200 +87.degree. 502.6 502 1305-1560.sup.3 50 F H (CH.sub.2).sub.2 CH.sub.3 H H H C(CH.sub.3).sub.3 -- 261-67 +69.degree. 548.7 548 1950-2100.sup.1 51 F H H (CH.sub.2).sub.2 CH.sub.3 H H C(CH.sub.3).sub.3 -- 255-59 +63.degree. 548.7 548 2145-2370 .sup.1 52 F H (CH.sub.2).sub.2 CH.sub.3 H CH.sub.3 H CH.sub.3 A 226-31 +101.degree. 520.6 520 1905-2175.sup.1 53 F H (CH.sub.2).sub.2 CH.sub. 3 H CH.sub.3 H CH.sub.3 B 232-38 +35.degree. 520.6 520 3300-3720.sup.1 54 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H CH.sub.3 A 176-88 +104.degree. 520.6 520 430-490.sup .2 55 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H CH.sub.3 B 214-19 +46.degree. 520.6 520 630-715.sup.2 56 F H H (CH.sub.2).sub.2 CH.sub.3 CH(CH.sub.3).sub.2 H CH.sub.3 A 133-35 +110.degree. 548.7 548 2100-2400.s up.1 57 F H H (CH.sub.2).sub.2 CH.sub.3 CH(CH.sub.3).sub.2 H CH.sub.3 B 210-12 +44.degree. 548.7 548 2850-3225.sup.1 58 F H H (CH.sub.2).sub.2 CH.sub.3 fenyl H CH.sub.3 A 235-40 +75.degree. 582.7 582 2100-2450.sup.1 59 F H H (CH.sub.2).sub.2 CH.sub.3 fenyl H CH.sub.3 B 157-82 +75.degree. 582.7 582 2760-3075.sup.1 60 H H (CH.sub.2).sub.2 CH.sub.3 H H H OC(CH.sub.3).sub.3 -- 140-42 +77.degree. 546.7 546 1500-1665.sup.1 61 H H H (CH.sub.2).sub.2 CH.sub.3 H H OC(CH.sub.3).sub.3 -- 160-65 +69.degree. 546.7 546 1620-1785.sup.1 62 F H (CH.sub.2).sub.2 CH.sub.3 H H H OC(CH.sub.3).sub.3 -- 171-73 +66.degree. 564.7 564 250-295.sup.4 63 F H H (CH.sub.2).sub.2 CH.sub.3 H H OC(CH.sub.3).sub.3 -- 161-64 +72.degree. 564.7 564 245-290.sup.4 64 F F H (CH.sub.2).sub.2 CH.sub.3 H H OCH.sub.2 CH.sub.3 -- 203-11 +99.degree. 554.6 554 325-370.sup.4 65 F F H (CH.sub.2).sub.2 CH.sub.3 H H OCH(CH.sub.3).sub.2 -- 196-209 +70.degree. 568.6 568 2235-2550.sup.1 66 H H (CH.sub.2).sub.2 CH.sub.3 H CH.sub.3 H OCH.sub.2 CH.sub.3 A + B 138-52 +102.degree. 532.6 532 300-370.sup.2 67 H H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 A + B 158-91 + 33.degree. 532.6 532 400-460.sup.2 68 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 A 196-98 +110.degree. 550.7 550 405-475.sup.2 69 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 B 212-14 +36.degree. 550.7 550 585-67.sup.2 70 F H (CH.sub.2).su b.2 CH.sub.3 H CH.sub.3 H OC(CH.sub.3).sub.3 A 154-57 +92.degree. 578.7 578 345-400.sup.2 71 F H (CH.sub.2).sub.2 CH.sub.3 H CH.sub.3 H OC(CH.sub.3).sub.3 B 161-68 +27.degree. 578.7 578 485-565.sup.2 72 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH(CH.sub.3).sub.2 A 221-24 +107.degree. 564.7 564 355-425.sup.2 73 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH(CH.sub.3).sub.2 B 212-15 +39.degree. 564.7 564 535-635.sup.2 74 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OC(CH.sub.3).sub.3 A 168-71 +103.degree. 578.7 578 485-570.sup.2 75 F H H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OC(CH.sub.3).sub.3 B 174-79 +31.degree. 578.7 578 255-310.sup.5 76 F H C(CH.sub.3).sub.3 H CH.sub.3 H OCH.sub.2 CH.sub.3 A 220-22 +95.degree. 564.7 564 380-430 77 F H C(CH.sub.3).sub.3 H CH.sub.3 H OCH.sub.2 CH.sub.3 B 227-37 +18.degree. 564.7 564 540-630.sup.2 78 F H H C(CH.sub.3).sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 A 229-32 +115.degree. 564.7 564 385-455.sup.2 79 F H H C(CH.sub.3).sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 B 246-51 +34.degree. 564.7 564 565-695.sup.2 80 F F (CH.sub.2).s ub.2 CH.sub.3 H CH.sub.3 H OCH.sub.2 CH.sub.3 A 167-70 +95.degree. 568.6 568 300-330.sup.5 81 F F (CH.sub.2).sub.2 CH.sub.3 H CH.sub.3 H OCH.sub.2 CH.sub.3 B 188-90 +26.degree. 568.6 568 365-395.sup.5 82 F F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 A + B 178-96 +68.degree. 568.6 568 3720-4155.sup.1 83 F F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 A 217-21 +105.degree. 568.6 568 290-340.sup.5 84 F F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH.sub.2 CH.sub.3 B 211-15 +32.degree. 568.6 568 341-395.sup.5 85 F F H (CH.sub.2 ).sub.2 CH.sub.3 CH.sub.3 H OCH(CH.sub.3).sub.2 A + B 198-210 +67.degree. 582.6 582 2190-3900.sup.1 86 F F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH(CH.sub.3).sub.2 A 232-37 +96.degree. 582.6 582 2190-2355.sup.1 87 F F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 H OCH(CH.sub.3).sub.2 B 225-32 +37.degree. 582.6 582 3630-3900.sup.1 88 F F H (CH.sub.2).sub.2 CH.sub.3 CH.sub.3 CH.sub.3 OCH.sub.2 .sup.1 On a Sephadex LH20 column (76 .times. 6.3 cm) using heptanechloroform-ethanol (20:20:1) as mobile phase .sup.2 On a Sephadex LH20 column (87.5 .times. 2.5 cm) using heptanechloroform-ethanol (20:20:1) as mobile phase .sup.3 On a Sephadex LH20 column (72 .times. 6.3 cm) using chloroform as mobile phase .sup.4 On a Sephadex LH20 column (80 .times. 2.5 cm) using chloroform as mobile phase .sup.5 On a Sephadex LH20 column (81.5 .times. 2.5 cm) using chloroform a mobile phase
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, OintmentSteroid, micronized 0.025 gLiquid paraffin 10.0 gWhite soft paraffin ad 100.0 gFormulation 2, OintmentSteroid 0.025 gPropylene glycol 5.0 gSorbitan sesquioleate 5.0 gLiquid paraffin 10.0 gWhite soft paraffin ad 100.0 gFormulation 3, Oil in water creamSteroid 0.025 gCetanol 5.0 gGlyceryl monostearate 5.0 gLiquid paraffin 10.0 gCetomacrogol 1000 2.0 gCitric acid 0.1 gSodium citrate 0.2 gPropylene glycol 35.0 gWater ad 100.0 gFormulation 4, Oil in water creamSteroid, micronized 0.025 gWhite soft paraffin 15.0 gLiquid paraffin 5.0 gCetanol 5.0 gSorbimacrogol stearate 2.0 gSorbitan monostearate 0.5 gSorbic acid 0.2 gCitric acid 0.1 gSodium citrate 0.2 gWater ad 100.0 gFormulation 5, Water in oil creamSteroid 0.025 gWhite soft paraffin 35.0 gLiquid paraffin 5.0 gSorbitan sesquioleate 5.0 gSorbic acid 0.2 gCitric acid 0.1 gSodium citrate 0.2 gWater ad 100.0 gFormulation 6, LotionSteroid 0.25 mgIsopropanol 0.5 mlCarboxyvinylpolymer 3 mgNaOH q.s.Water ad 1.0 gFormulation 7, Suspension for injectionSteroid, micronized 0.05-10 mgSodium carboxymethylcellulose 7 mgNaCl 7 mgPolyoxyethylene (20) sorbitan 0.5 mgmonoleatePhenyl carbinol 8 mgWater, sterile ad 1.0 mlFormulation 8, Aerosol for oral and nasal inhalationSteroid, micronized 0.1% w/wSorbitan trioleate 0.7% w/wTrichlorofluoromethane 24.8% w/wDichlorotetrafluoromethane 24.8% w/wDichlorodifluoromethane 49.6% w/wFormulation 9, Solution for atomizationSteroid 7.0 mgPropylene glycol 5.0 gWater ad 10.0 gFormulation 10, Powder for inhalationA gelatin capsule is filled with a mixture ofSteroid, micronized 0.1 mgLactose 20 mgThe powder is inhaled by means of an inhalation device.______________________________________
PHARMACOLOGY
The affinity of the new androstane-17.beta.-carboxylic acid esters to the glucocorticoid receptor
All steroids according to the present invention are physiologically active compounds. The affinity of the novel androstane-17.beta.-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]-16.alpha.,17.alpha.-butylidenedioxy-11.beta.,21-dihydroxypregna-1,4-diene-3,20-dione) a highly active glucocorticoid with a favourable ratio between local and systemic effects (Thalen 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 NaMoO.sub.4, 10 mM mercaptoethanol. The homogenate was centrifuged for 15 min at 20,000.times.g. Portions of the 20,000.times.g supernatant (230 .mu.l) were incubated for about 24 h at 0.degree. C. with 100 .mu.l phenylmethylsulphonylfluoride (an esterase inhibitor, final conc. 0.5 mM). 20 .mu.l unlabelled competitor and 50 .mu.l .sup.3 H-labelled dexamethasone (final conc. 3 mM). Bound and free steroid were separated by incubating the mixture with 60 .mu.l 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 NaMoO.sub.4 for 10 min at 0.degree. C. Following a centrifugation at 500.times.g for 10 min, 230 .mu.l or the supernatant was counted in 10 ml Insta-Gel in a Packard scintillation spectrophotometer. The supernatants were incubated with (a) [.sup.3 H]dexamethasone alone, (b) [.sup.3 H]dexamethasone plus 1000 fold excess of unlabelled dexamethasone and (c) [.sup.3 H]dexamethasone plus 0.03-300 fold "excess" of competitor. The nonspecific binding was determined when 1000 fold excess of unlabelled dexamethasone was added to [.sup.3 H]-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 4______________________________________Table summarizing relative binding affinities (RBA) to theglucocorticoid receptor of some of the investigated compounds.Compound accordingto Ex. No. RBA______________________________________Budesonide 1 4 epimer B 0.30 5 epimer B 0.1727 0.5038 0.0455 0.2064 0.0567 0.0469 0.4484 1.0387 0.63______________________________________

Number	Name	Date	Kind
3828080	Phillipps et al.	Aug 1974
3981894	Phillipps et al.	Sep 1976

Number	Date	Country
3126732	Mar 1982	DEX
3149475	Jul 1982	DEX
378109	Nov 1973	SEX
396079	Jan 1974	SEX
436572	Aug 1977	SEX

16,17-acetalsubstituted androstane-17.beta.-carboxylic acid esters prossessing high binding affinity to the glucocorticosteroid receptor

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