NO864979L - ANDROSTAN-17 BETA CARBOXYLIC ACID COMPOUNDS. - Google Patents

Description

The present invention relates to new androstane-17|3-carboxylic acid compounds. These can be used for the production of anti-inflammatory and anti-allergic glucocorticosteroids, namely androstane-17(3-carboxylic acid esters.

The glucocorticosteroids are one of the most effective and widely used classes of anti-inflammatory agents known. They are very effective in preventing or reducing the severity of a wide range of inflammatory, immunological and allergic diseases in the respiratory tract (e.g. asthma, rhinitis), in the skin (e.g. eczema, psoriasis) or in the intestines ( eg ulcerative colitis, Morbus Crohn). However, there is a general desire to minimize the systemic side effects. One way of doing this involves placing the steroid locally on the organ to be treated, e.g. the respiratory tract, thereby allowing lower systemic concentrations of the steroid. A quick inactivation e.g. by hydrolysis in the target organ or in the general circulation will further reduce the systemic side effects.

The steroid compounds in the present invention are compounds with the general formula:

where

X 1 represents a hydrogen, chlorine, bromine or fluorine atom;

X 2 represents a hydrogen, chlorine, bromine or fluorine atom;

Rj represents a p-hydroxy group, an e-chloro atom or an oxo group;

R 2 represents a hydrogen atom, a methylene group or a cx- or 3-methyl group;

R 3 represents a hydrogen atom or an acyl group with 1-8 carbon atoms;

R 4 represents a hydrogen atom, a (C 1 -C 5 )-alkyl group or a

phenyl group;

R 5 represents a hydrogen atom, a (C 1 -C 5 )-alkyl group or a

phenyl group;

Y represents either CRyRg, O, S or NR9, where R7, Rg and R9 are selected from hydrogen or from straight or branched hydrocarbon chains with 1-8 carbon atoms or from a phenyl group;

R(5represents a hydrogen atom; a methyl group; a phenyl or an alkenyl or cycloalkenyl group optionally substituted with alkyl, nitro, carboxy, alkoxy, halogen, cyano, caralkyloxy and trifluoromethyl group(s); a (Cj -C5)-alkyl group substituted with at least one halogen atom; a saturated or unsaturated carbocyclic or heterocyclic (0, S, N) ring system containing 3-10 atoms in the ring system; a C1-alkyl group substituted with either one or two alicyclic or aromatic 3-, 4-, 5- or 6-membered ring system(s) or one, two or three straight or branched, saturated or unsaturated

hydrocarbon chains of 1-18 carbon atoms; and

represents a single or double bond.

In general, R 5 in formula I is an alkyl group with 1-5 carbon atoms, preferably a methyl, ethyl, propyl, allyl, isopropyl, metallyl, isobutyl, cyclopropylmethyl, cyclobutyl or cyclopentyl group.

The halogen atoms for positions X 1 and X 2 are preferably a fluorine or chlorine atom; the acyl group (R3) is preferably an acetyl, propionyl, butyryl or valeroyl group. R^ usually represents a 3-hydroxy group and _■; generally represents a double bond.

Of the subgroup

those compounds in which R 4 = H and R 5 = methyl or R 4 = methyl and R 5 = H are preferred.

A preferred class of compound of formula I which has particularly good pharmacological activity includes the following esters of 9cx-fluoro-ll3-hydroxy-16 3-methyl-3-oxo-17oc-propionylox y-andros ta-1, 4-diene-173-carboxylic acid and 9a-chloro-113-hydroxy-163-methyl-3-oxo-17c-propionyl-oxy-androsta-1,4-diene-173-carboxylic acid: 1' - ethoxycarbonyloxyethyl, 1' - isopropoxycarbonyloxyethyl, 1'-propoxycarbonyloxyethyl, 1'-cyclopropylmethoxycarbonyloxyethyl, 1'-cyclobutoxycarbonyloxyethyl, 1'-isobutoxycarbonyloxyethyl, 1'-cyclopentoxycarbonyloxyethyl, r-(2-chloroethoxy)carbonyloxyethyl and 1'-acetoxyethyl.

Manufacturing methods

Epimers resulting from the asymmetric center in the ester group with formula I constitute a further feature of the invention and the invention includes the production of such epimers as well as mixtures thereof. The epimers have practically identical solubility properties. Consequently, they have been impossible to separate and isolate from the epimeric mixture by conventional methods for resolving stereoisomers, e.g. fractional crystallization. However, the epimers can be separated in view of different mobility on the stationary phase in a chromatographic system. The separation can be carried out e.g. on silicon dioxide or especially on cross-linked dextrins of the "Sephadex ^ LH" type, e.g. "Sephadex R LH-20" with a suitable organic solvent as eluent. As eluent on a "Sephadex ^ LH-20" column, a halogenated hydrocarbon such as chloroform or a mixture of heptane-chloroform-ethanol in the ratio of 0-50:50-100:10-1 has been used successfully, preferably a 20:20:1 mix. In these straight-phase chromatography systems, the epimers have been arbitrarily designated A and B, respectively, in order of elution from the column.

Source material

The 173-carboxylic acid starting materials are prepared by removal of the 21-carbon atom from an appropriate 21-hydroxy-3,20-dioxopregn-4-ene or pregna-1,4-diene. This is easily achieved by any known method such as the use of sodium hypobromate, sodium bismuthate, sodium periodate or oxygen (air) in alkaline solution. The oxidation is preferably carried out with periodic acid in a solvent medium such as tetrahydrofuran/H 2 O and preferably at room temperature. Suitable 21-hydroxy-3,20-dioxopregn-4-enes or pregna-1,4-dienes include known compounds such as betamethasone, dexamethasone, paramethasone, beclomethasone, flumethasone and the like. By means of the following methods which are generally known in the art, steroids of relatively simple structure can be converted into other structures as desired.

The esterification of the 17cx-hydroxy group in the preparation of the new androstane compounds can be carried out in a known manner, e.g. by reacting the 17oc-hydroxy parent compound in a suitable carboxylic acid or a reactive derivative thereof, such as an acid anhydride, acid halide or orthoester in the presence of a suitable acid catalyst and a solvent at temperatures of 20-100° C. Suitable carboxylic acids and reactive derivatives include e.g. acetic acid, propionic acid, butyric acid, etc. and the corresponding acid anhydrides and acid halides and orthoesters. Solvents include non-hydroxyl solvents such as methylene chloride, chloroform, benzene and the like, while suitable acid catalysts include p-toluenesulfonic acid, sulfosalicylic acid, perchloric acid, strongly acidic cation exchange resins and the like.

For the preparation of the 17a-esters of the 173-carboxylic acids that can be used in the preparation of the compounds of the present invention, it is often preferred to treat the 17a-hydroxy-17e-carboxylic acid parent compound with an appropriate acid anhydride or acid halide to obtain the mixed anhydride of androstane The -173-carboxylic acid and the carboxylic acid ester of the acid anhydride or acid halide starting compound, this reaction being conveniently carried out at an elevated temperature and the resulting anhydride then being solvolysed under acidic conditions (e.g. using aqueous acetic acid) or under basic conditions (eg aqueous pyridine or a secondary amine such as diethylamine in acetone).

End connections

The compounds of the present invention can be obtained by any of the following methods.

The esters can be prepared by reacting the 17p-carboxylic acid in the form of a salt, e.g. an alkali metal salt or a tetraethylammonium or tetrabutylammonium salt with a halogen ester of the formula:

where Y, R 1 , R 4 , R 5 have the same meaning as stated above and Z is a halogen atom, preferably a chlorine or bromine atom, or a functionally equivalent group such as a sulfonyloxy radical. The reaction is carried out in an inert organic solvent, e.g. acetone, methyl ethyl ketone, dimethyl formamide, dimethyl sulfoxide, methylene chloride or chloroform, and at temperatures between 0 and 100 "C. The reaction can also be carried out in the presence of a crown ether.

Pharmaceutical preparations

The compounds in the present invention can be used for different types of local administration depending on the site of inflammation, e.g. percutaneously, parenterally or for local administration in the respiratory tract by inhalation. An important aim with the type of formulation is to achieve optimal bioavailability for the active steroid ingredient. For percutaneous preparations, this is advantageously achieved if the steroid compound is dissolved with a high thermodynamic activity in the carrier. This is achieved by using a suitable system of solvents comprising suitable glycols, such as propylene glycol or 1,3-butanediol either as such or in combination with water.

It is also possible to dissolve the steroid compound either completely or partially in a lipophilic phase by means of a surfactant as a solubilizing agent. Percutaneous preparations can be an ointment, an oil-in-water cream, a water-in-oil cream or a lotion. In the emulsion carriers, the system including the dissolved active component can constitute the disperse phase as well as the continuous phase. The steroid compound can also exist in the above-mentioned preparations as a micronized, solid substance.

Pressurized aerosols for steroid compounds are intended for oral or nasal inhalation. The aerosol system is constructed in such a way that each delivered dose contains 10-1000 µg, preferably 20-250 µg, of the active steroid compound. The most active steroids are administered in the lower part of the dose range. The micronized steroid compound consists of particles substantially smaller than 5 µm which are suspended in a propellant mixture by means of a dispersing agent such as sorbitan trioleate, oleic acid, lecithin or sodium salt of dioctylsulfuric acid.

Execution examples

The invention is further illustrated by the following examples. All mass spectra have been obtained by chemical ionization mass spectrometry and they are all consistent with the molecular weights of the compounds. The purity of each epimer has been determined in a High Performance Liquid Chromatography (HPLC) system using a "pBondapak (^" column (300 x 3.9 mm i.d.) at a flow rate of 1.0 ml/min .and with a ratio of ethanol to water between 35:65 and 65:35 as mobile phase.

Examples

The compounds listed in Table 1 were prepared, isolated and purified in a manner analogous to the method described in Examples 1-5.

Example 1

Preparation of 1' - ethoxycarbonyloxyethyl- 9c< - fluoro- 11P - hydroxy- 16p - methyl- 3- oxo- 17cx- propionyloxy- androstal, 4- diene- 17p- carboxylate

A mixture of betamethasone (4.0 g) in tetrahydrofuran (45 ml) and periodic acid (7.0 g) dissolved in water (25 ml) is stirred at room temperature for 2 hours. Water (40 ml) is added and the organic solvent is removed under reduced pressure. The resulting crystalline precipitate is collected by filtration, washed with water and dried to obtain 90-fluoro-113,17c-dihydro-163-methyl-3-oxo-androsta-1,4-diene-176p<->carboxylic acid (4, 0g).

The acid is suspended in methylene chloride (80 ml) and triethylamine (4.1 ml) and propionyl chloride (3.5 ml) are added at 0°C. After stirring at 0°C for 80 minutes, the reaction mixture is diluted with methylene chloride and washed successively with 3% sodium bicarbonate, 2N hydrochloric acid and water, and then dried and evaporated in vacuo.

The solid is treated with diethylamine (3.0 mL) in acetone (130 mL) for 90 minutes at room temperature. After evaporation of the organic solvent, water is added and the mixture is extracted with ethyl acetate. Acidification with 2N hydrochloric acid and extraction with ethyl acetate gives, after the usual work-up, 9a-fluoro-lle-hydroxy-16p-methyl-3-oxo-17a-propionyloxyandrosta-1,4-diene-17p-carboxylic acid as a white crystalline substance (3, 8g).

Modification 1

A mixture of 9α-fluoro-113,17α-dihydroxy-163-methyl-3-oxo-androsta-1,4-diene-17e-carboxylic acid (2.5 g), potassium bicarbonate (800 mg), 18-crown-6 - (10.5 mg) and cx-chloroethyl ethyl carbonate (1.2 g) in dimethylformamide (50 ml) are stirred at 80° C. for 3 hours. The reaction mixture is diluted with 10% sodium chloride (200 ml) and extracted with methylene chloride (3 x 100 ml). The organic phase is washed successively with 5% sodium bicarbonate (100 ml) and water (3 x 75 ml), dried and evaporated in vacuo.

Chromatography on "Sephadex ^ LH-20" with chloroform gives the epimeric mixture of the title compound.

A further chromatographic step on "Sephadex ^ LH-20" with heptane-chloroform-ethanol (20:20:1) gives the pure epimers with good purity as determined by HPLC analysis.

Epimer A: 1.14 g (72%) m.p. 187-190°C [cx]D<5>= +86.7°

(c = 0.2, CH2Cl2)

HPLC analysis of epimer A: 99.3%

MS-Cl (CH 4 ): MH<+>= 551; M<+>+ 29 = 579

Epimer B: 1.27 g (80%) m.p. 218-221 °C [c<]D<5>= +0.9°

(c = 0.2, CH2Cl2)

HPLC analysis of epimer B: 99.2%

MS-Cl (CH 4 ): MH<+>= 551; M<+>+ 29 = 579

Modification 2

By carrying out the esterification step at 80°C for 3 hours, but without the presence of 18-crown-6, a yield of 84% of the epimeric mixture of the title compound was obtained. Otherwise, the conditions were identical to those in method 1.

Modification 3

A mixture of 9α-fluoro-113-hydroxy-16β-methyl-3-oxo-17α-propionyl-oxyandrosta-1,4-diene-17β-carboxylic acid (435 mg), potassium bicarbonate (111 mg) and α-bromomethylethyl carbonate (209 mg) in acetonitrile (50 ml), stirred at 40°C for 5 hours. The solvent is evaporated in vacuo and the residue is dissolved in methylene chloride. The organic phase is washed successively with 5% sodium bicarbonate and water, dried and evaporated in vacuo. Chromatographic work-up which in method 1 gives the pure epimers A (155 mg) and B (170 mg) of 1'-ethoxycarbonyloxyethyl-9c<-fluoro-lle-hydroxy-16e-methyl-3-oxo-17a-propionyloxyandrosta-l, 4-diene-173-carboxylate.

The reaction can also be carried out in any aprotic solvent such as dimethylformamide or dimethylsulfoxide.

Modification 4

A mixture of 9a-fluoro-11g-hydroxy-16p-methyl-3-oxo-17c<-propionyl-oxyandrosta-1,4-diene-17e-carboxylic acid (250 mg), potassium bicarbonate (69 mg), a-chloroethyl ethyl carbonate ( 102 mg) and lithium bromide (240 mg) in acetone (25 mL), was refluxed for 17 h. Using the work-up method in method 1 gave the epimeric mixture of 1'-ethoxycarbonyloxyethyl-9a-fluoro-lle-hydroxy-16e-methyl-3-oxo-17a-propionyloxyandrosta-1,4-diene-17e-carboxylate (20 mg ).

When a mixture of α-chloroethyl ethyl carbonate (1.5 g) and lithium bromide (3.4 g) was refluxed in acetone (20 mL) for 2 h, an oil (0.8 g) of α-chloroethyl ethyl carbonate (58%) and α-bromomethylethyl carbonate (42%) obtained.

Modification 5

To the potassium salt of 9a-fluoro-113-hydroxy-16e-methyl-3-oxo-17a-propionyloxyandrosta-1,4-diene-17a-carboxylic acid (475 mg) in methylene chloride (20 ml) and water (10 ml) was added tetrabutylammonium hydrogen sulfate (340 mg) was added while stirring. The pH was adjusted to 7 with 2M sodium hydroxide. The organic phase was evaporated. The residue was dissolved in acetone (50 ml) and α-chloroethyl ethyl carbonate (150 mg) was added. The solution was stirred for 4 hours at 50 °C. The acetone was removed by vacuum and the residue was extracted with n-butyl acetate, the organic phase was washed with 3% sodium bicarbonate and twice with water, dried and evaporated. Use of the same chromatographic method as in method 1

gave the pure epimers (A: 81 mg; b: 83 mg) of the title compound.

Modification 6

To a solution of 9a-fluoro-113-hydroxy-16e-methyl-3-oxo-17a-propionyloxyandrosta-1,4-diene-17e-carboxylic acid (451 mg), 1,5-diaza-bicyclo[5.4.0] undecene-5 (152 mg) in benzene (10 ml), α-chloroethyl ethyl carbonate (152 mg) in benzene (5 ml) was added. The solution was stirred under reflux for 6 hours and then overnight at room temperature. The solvent was removed under vacuum and the residue dissolved in methylene chloride, washed with sodium bicarbonate and twice with water, dried and evaporated. Using the same chromatographic method as in method 1 gave the pure epimers (A: 48 mg; B: 55 mg) of the title compound.

Example 2

Preparation of 1' - isopropylcarbamoyloxyethyl- 9c< - fluoro- 11 3 - hydroxy- 16p - methyl- 3- oxo- 17a- propionyloxyandrosta- 1, 4- diene- 173- carboxylate Treatment of 9a-fluoro-ll3-hydroxy-163- methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carboxylic acid (1.4 g) with potassium bicarbonate (450 mg) and T-chloroethylisopropyl carbamate (695 mg, prepared from α-chloroethyl chloroformate and isopropylamine in diethyl ether ) in dimethylformamide (30 mL) at 80°C

for 3 hours by following the same preparation method as in example 1,

gave epimer A (110 mg; mp 210.214°C; HPLC: 99.7%, NMR (<!>H); 6.69 ppm (quartet, C-CH(CH3)-0-); MS-CI ( CH4): MH<+>: 564, M<+>+ 29 = 592) and epimer B (28 mg, mp 183-186° Cc: HPLC: 99.1%, NMR (<!>H): 6 .81 ppm (quartet, -0-CH(CH3)-0-): MS-CI (CH4): MH<+>: 564, M<+>+ 29 = 592) of 1'-i sopr opylka rbamoyloxyethyl - 9α-Fluoro-1α-hydroxy-16-3-methyl-3-oxo-17α-propionyloxyandrosta-1,4-diene-173-carboxylate.

Example 3

Preparation of r- diethylcarbamoyloxymethyl- 9a- fluoro- lle- hydroxy- 16g-methyl- 3- oxo- 17a- propionyloxyandrosta- 1, 4-diene- 173- carboxylate Conversion of 9a-fluoro-113-hydroxy-163-methyl-3 -oxo-17a-propionyloxy-androsta-1,4-diene-173-carboxylic acid (1.1 g) with potassium bicarbonate (10 g) and r-chloroethyl diethyl carbamate (825 mg; prepared from a-chloroethyl chloroformate and diethylamine in diethyl ether) in dimethylformamide (20 ml) at room temperature for 48 hours following the same work-up method as in Example 1 gave epimer A (120 mg; mp 184-187° C; HPLC: 99.8%, NMR (<1>H): 6.71 ppm (quartet, -0-CH(CH3)-0-); MS-CI (CH4): MH2+: 606, M<+>+ 29 = 634) and epimer B (104 mg, mp 156- 160°C; HPLC: 99.8%; NMR (1H) : 6.81 = 634) of r-diethylcarbamoyloxyethyl-9α-fluoro-113-hydroxy-163-methyl-3-. oxo-17α-propionyloxyandrosta-1,4-diene-173-carboxylate.

Example 4

Preparation of r- acetyloxyethyl- 9a- fluoro- ll3- hydroxy- 163- methyl- 3- oxo-17a- propionyloxyandrosta- 1, 4- diene- 173- carboxylate

Treatment of 9a-fluoro-113-hydroxy-163-methyl-3-oxo-17a-propionyloxy-androsta-1,4-diene-173-carboxylic acid (1.2 g) with potassium bicarbonate (1.2 g) and a- chloroethyl acetate (1.2 g; prepared from acetyl chloride, paraldehyde and zinc chloride under nitrogen at -10 °C for 2 h) in dimethylformamide (40 ml) at 50 °C for 20 h following the same work-up method as in Example 1 gave the epimer A (391 mg; HPLC: 99.7%, NMR ( 1U) : 6.79 ppm (quartet, -O-CH(CH3)-0-); MS-Cl (CH4): MH<+>: 521, M<+>+ 29 = 549) and epimer B (393 mg; mp 211-212° C; HPLC: 99.8%; NMR ^H): 6.93 ppm (quartet, -O-CH(CH3) -0-); MS-Cl (CH 4 ): MH<+>: 521, M<+>+ 29 = 549) of 1'-acetyl oxyethyl 1-9cx-fluor-lle-hydroxy-163-methyl-3-oxo- 17a-propionyl-oxyandrosta-1,4-diene-17e-carboxylate.

Example 5

Preparation of 1'-isopropylthiocarbonyloxyethyl-9cx-fluoro-11e-hydroxy-16g-methyl-3-oxo-17a-propionyloxyandrostal, 4-diene-17e-carboxylate. Treatment of 9a-fluoro-113-hydroxy-16g-methyl-3-oxo-17a-propionyloxy-androsta-1,4-diene-17g<->carboxylic acid (1.0 g) with potassium bicarbonate (350 mg) and a- Chloroethyl-5-isopropylthiocarbonate (600 mg; prepared from α-chloroethyl chloroformate and 2-propanethiol (in pyridine and diethyl ether) in dimethylformamide (20 mL) at room temperature for 48 h following the same work-up method as in Example 1 gave epimer A (14 mg ; NMR (^H): 6.92 ppm (quartet, -0-CH(CH3)-0-); MS-Cl (CH4): MH<+>= 581; M<+>+ 29 = 609) and epimer B (18 mg, NMR ^ U) : 6.98 ppm (quartet, -0-CH(CH3)-0-); MS-CI (CH4): MH<+>= 581) r-isopropylthiocarbonyloxyethyl-9a- fluoro-llp-hydroxy-16b-methyl-3-oxo-17a-propionyloxyandrosta-1,4-diene-17e-carboxylate.

Example 34

Pharmaceutical preparations

The following examples illustrate preparations intended for different topical forms of administration. The amount of active steroid in the percutaneous preparations is usually 0.001-0.2% (w/w), preferably 0.0-0.1%

(weight/weight).

The powder is inhaled using an inhalation device.

F a r_m akplogy

The affinity of the new androstane-17e-carboxylic acid esters for the gluco-corticoid receptor

All steroids in the present invention are physiologically active compounds. The affinity of the new androstane-17e-carboxylic acid esters for the glucocorticoid receptor has been used as a model for determining the degree of anti-inflammatory activity. Their receptor affinities have been compared with budesonide ([22R,S]-16oc,17cx-butylidenedioxide-1<p>,21-dihydroxypregna-1,4-dien-3,20-dione) a highly active glucocorticoid with an advantageous relationship between local and systemic effects (Thalén and Brattsand, Arzneim.-Forsch. 29, 1687-90 (1979)).

Male Sprague-Dawley rats, one to two months old, were used throughout the study. The thymus was removed and placed in 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-4, 10 mM mercaptoethanol. The homogenate was centrifuged for 15 minutes at 20,000 x g. Portions of the 20,000 x g supernatant (230 µl) were incubated for approx. 24 hours at 0°C with 100 µl phenylmethylsulfonyl fluoride (an esterase inhibitor, final conc. 0.5 mM), 20 µl unlabeled competitor and 50 µl ^H-labeled dexamethasone (final conc. 3 nM). Bound and free steroid were separated by incubating the mixture with 60 µl of 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 4 in 10 min at 0°C. After a centrifugation at 500 x g for 10 minutes, 230 µl of the supernatant was subjected to counting in 10 ml of Insta-Gel in a Packard scintillation spectrophotometer. The supernatants were incubated with a) [^H]dexamethasone alone, b) [^H]dexamethasone plus 1000-fold excess of unlabeled dexamethasone and c) [^H]dexamethasone plus 0.03-300-fold "excess" of competitor. The non-specific binding was determined when 1000-fold excess of unlabeled dexamethasone was added to [<3>H]-labeled 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 percent specific binding of labeled dexamethasone. For each concentration of a competitor, the percentage specifically bound radioactivity is plotted against the log of the concentration of competitor. The curves are compared at the 50% specific binding level and are referenced to budesonide, which is given a relative binding affinity (RBA) equal to 1.