The present invention relates to a method for the preparation of Trilostane.

Trilostane ((4α,5α,17β)-4,5-epoxy-3,17-dihydroxyandrost-2-ene-2-carb onitrile; 2) is used in human and veterinary medicine to treat endocrine disorders. Its primary indication is as an anticancer agent but it is also used in the treatment of Cushing's disease and other hyperadrenocortical conditions.

Trilostane is produced using multi-step syntheses. A typical example of the final step in a synthesis of Trilostane is shown in reaction scheme 1. This step involves the conversion of (4α,5α,17β)-4,5-epoxyandrost-2-eno(2,3-d)isoxazol-17-ol (1) to Trilostane (2). A base (methoxide ion) is used to remove the labile isoxazole proton which causes the (2,3- d)isoxazole ring to open and produce an enolate which is then acidified using aqueous acid to yield Trilostane (2).

Reaction Scheme 1

Following production of Trilostane (2) it is necessary to carry out at least one recrystallisation to obtain a final product of satisfactory purity. Having to recrystallise the product is clearly undesirable since it further complicates the overall synthetic process and is likely to reduce significantly the final product yield obtained.

Neumann et al.x describe the use of sodium methoxide in tetramethylurea to effect an isoxazole ring-opening reaction to yield Trilostane followed by recrystallisation from dimethylformamide. A crude product yield of 97.4 % was obtained but, following recrystallisation, the purified product yield was only 56.0 %. A further problem with this method is that because dimethylformamide has a relatively high boiling point (153 °C) an undesirable level of dimethylformamide is likely to be retained in the recrystallised product.

Neumann et al) appear to have tested one or more hydroxylic solvents (alcohols) as alternative recrystallisation solvents. However, poor yields were obtained which the authors presume was due to decomposition of the 2-cyano-3-keto product with the evolution of toxic hydrogen cyanide since, "an HCN-type odor was usually noted". The decomposition products were not investigated further but it was noted from TLC studies that the 2-cyano-3-keto spots had almost completely disappeared and several new spots had appeared. This work indicates that hydroxylic solvents are unsuitable for use in the final step in the synthesis of Trilostane because they degrade the intended reaction product such that final product yields are significantly reduced and are liable to generate highly toxic hydrogen cyanide.

Further processes for the production of 2-cyano-3-keto-steroids have been described. US 3,135,743 discloses the preparation of a 2-cyano-3-keto-steroid involving an isoxazole ring- opening step similar to that of reaction scheme 1 in which sodium methoxide is used in ether to effect the ring-opening step. The final product is then recrystallised twice from an ether- hexane mixture. US 3,296,255 discloses the preparation of a series of 2-cyano-3-keto-steroids from isoxazole precursors. In this case the isoxazole ring-opening step is carried out using sodium methoxide or sodium ethoxide in diethyl ether or tetrahydrofuran and a number of different recrystallisation solvents then used, e.g. methyl ethyl ketone, or mixtures of tetrahydrofuran-ethyl acetate or pyridine-dioxane. GB 1,123,770 describes the synthesis of a 2-cyano-4,5-epoxy androstane steroid in which the isoxazole ring of the precursor is cleaved using sodium methoxide in tetrahydrofuran. A pyridine-dioxane mixture is then used as a recrystallisation solvent to purify the final product.

An object of the present invention is to obviate or mitigate one or more of the aforementioned problems to provide an improved method for the preparation of Trilostane.

According to a first aspect of the present invention there is provided a method for the preparation of Trilostane from (4α,5α,17β)-4,5-epoxyandrost-2-eno(2,3-d)isoxazol-17-ol as a precursor compound, the method comprising the steps of: a) dissolving the precursor compound in methanol; b) treating the precursor compound with a base to effect cleavage of the (2,3-d)isoxazole ring of the precursor compound; c) adding acid and water to the product of step b) to obtain a precipitate of Trilostane; and d) isolating the precipitate of Trilostane.

Preparing Trilostane in accordance with this aspect of the invention enables a high yield of a satisfactorily pure product to be obtained without the need to carry out one or more recrystallisations. This represents a major improvement in the preparation of this drug since it enables Trilostane to be produced with significantly greater efficiency than using current methods. Not only is it surprising that carrying out the ring-opening reaction in methanol negates the need to recrystallise the final product, the fact that methanol can be used at all in the final step in the production of Trilostane was very unexpected in view of the observations of Neumann et al} which strongly suggested that solvents such as methanol would be highly unsuitable for this purpose. Additionally, since methanol has a much lower boiling point (65 0C) than, for example, dimethylformamide, problems related to residual solvent remaining in the final product are greatly reduced.

Any suitable base may be used in this aspect of the invention provided it is sufficiently basic to remove the labile isoxazole proton so as to initiate cleavage of the isoxazole ring of the precursor compound when the precursor compound is dissolved in methanol. Preferably the base is an alkali metal hydroxide. More preferably the base is selected from the group consisting of sodium hydroxide and potassium hydroxide. Alternatively, the base may be an alkali metal alkoxide, e.g. methoxide, ethoxide or the like. A particularly preferred alkali metal alkoxide may be selected from the group consisting of: sodium methoxide and potassium methoxide.

It is preferred that cleavage of the (2,3-d)isoxazole ring of the precursor compound is effected using at least one mole equivalent of the base compared to the precursor compound. Preferably cleavage of the (2,3-d)isoxazole ring of the precursor compound is effected using 1.00 to 1.50 mole equivalents of the base compared to the precursor compound. More preferably cleavage of the (2,3-d)isoxazole ring of the precursor compound is effected using 1.05 to 1.30 mole equivalents of the base compared to the precursor compound. It is particularly preferred that more than one mole equivalent of base compared to the precursor compound is used so that the excess base not required to effect cleavage of the isoxazole ring can react with any acidic contaminants which may be present. While cleavage of the isoxazole ring may be effected at room temperature or below given a sufficiently long reaction time, in a preferred embodiment of this aspect of the invention cleavage of the isoxazole ring is effected at an elevated temperature, such as a temperature at which the precursor can be treated with base under reflux conditions. Preferably cleavage of the (2,3-d)isoxazole ring of the precursor compound is effected at a temperature in the range 15 0C to a temperature at which the precursor can be treated with base under reflux conditions. More preferably cleavage of the (2,3-d)isoxazole ring of the precursor compound is effected at a temperature in the range 30 to 50 °C. Yet more preferably cleavage of the (2,3- d)isoxazole ring of the precursor compound is effected at a temperature in the range 40 to 45 0C.

Preferably the precursor compound is treated with the base over a time period of up to 4 hours. More preferably the precursor compound is treated with the base over a time period of 2 to 3 hours. Most preferably the precursor compound is treated with the base over a time period of approximately 2 hours.

In order to obtain a precipitate of Trilostane the product of step b) of this aspect of the invention is acidified using acid and water. It is preferred that the acid is added to the product of step b) before the addition of the water. Further preferred embodiments of this aspect of the invention comprise the addition of the acid to the product of step b) during or after the addition of the water. The acid may be an aqueous acid. If an aqueous acid is used it may or may not be necessary to add additional water before, during or after addition of the aqueous acid.

Whilst any appropriate acid may be used in this aspect of the invention, an important factor in deciding upon a suitable acid is the ease with which it can be removed from the Trilostane product. This is partly determined by the relative solubility of the salt form of the acid in water compared to its solubility in methanol. A salt of the acid with a higher relative aqueous solubility will be preferentially retained in the aqueous phase of the reaction mixture to a greater extent than a salt form of the acid with a lower relative aqueous solubility. Thus, a salt of the acid with a higher relative aqueous solubility can be more readily separated from the Trilostane product which will be retained in the methanolic phase. Preferably the acid is an organic acid, such as a carboxylic acid. The carboxylic acid may be selected from the group consisting of formic acid, acetic acid, propanoic acid and butanoic acid. Acetic acid is preferred because of the high relative aqueous solubility of certain preferred salts of the acetate ion. Alternatively, a mineral acid may be used, for example, the mineral acid may be selected from the group consisting of hydrochloric acid and sulphuric acid.

It is preferred that 1.15 to 1.30 mole equivalents of the acid compared to the base is added to the product of step b). 1.20 to 1.25 mole equivalents of the acid compared to the base may be added to the product of step b).

In a preferred embodiment of this aspect of the invention the product of step b) is maintained at a temperature in the range 15 0C to a temperature at which the precursor can be treated with base under reflux conditions during addition of the acid and water. Preferably the product of step b) is maintained at a temperature of 30 to 50 °C, more preferably 40 to 45 0C, during addition of the acid and water.

Isolation of the precipitate of Trilostane is preferably carried out by filtering the product of step c). It is preferred that the product of step c) is maintained at a temperature in the range 15 to 25 °C during filtration.

The first aspect of the present invention is directed to the preparation of Trilostane using methanol as solvent, which is in contrast to the teaching of Neumann et al1. As stated above, the use of methanol in this way provides a number of important advantages over current methods. It has been found, however, that solvents other than methanol may also be used. Thus, according to a second aspect of the present invention there is provided a method for the preparation of Trilostane from (4α,5α,17β)-4,5-epoxyandrost-2-eno(2,3-d)isoxazol-17-ol as a precursor compound, the method comprising the steps of: a) dissolving the precursor compound in a solvent; b) treating the precursor compound with a base to effect cleavage of the (2,3-d)isoxazole ring of the precursor compound; c) adding acid and water to the product of step b) to obtain a precipitate of Trilostane; and d) isolating the precipitate of Trilostane. It is preferred that when using a method in accordance with the second aspect of the present invention the solvent does not cause decomposition of Trilostane or the precursor compound. The solvent may be an alcohol or an ether. Preferred alcohols include lower alcohols such as methanol, ethanol, isopropanol and the like. Preferred ethers include tetrahydrofuran and the like.

The invention is illustrated with reference to the following non-limiting Example. EXAMPLE

The following procedure may be used for the conversion of (4α,5α,17β)-4,5-epoxyandrost-2- eno(2,3-d)isoxazol-17-ol (1) to Trilostane ((4α,5α,17β)-4,5-epoxy-3,17-dihydroxyandrost-2- ene-2-carbonitrile; 2).

(1) (2) Inventory

Process

1. Charge compound (1) (10.0 g, 30.4 mmols, 1.0 equiv) to a round-bottomed flask fitted with a stirrer. 2. Charge methanol (150 ml) and sodium hydroxide (1.49 g, 37.3 mmols, 1.23 equivs) to the flask and leave to stir-out. 3. Leave the solution to stir-out with warming (40 - 45 °C, two hours). 4. Hold the temperature of the stirred reaction mixture (40 - 45 °C) and slowly charge acetic acid (2.74 g) over two-hours. 5. Add water in minimum time consistent with maintaining the temperature of the resulting slurry at 40 - 45 °C. 6. Leave the slurry to stir-out and cool to 18 - 20 °C. 7. Collect the solid by filtration and wash with water. 8. Charge filter cake to a vacuum oven to dry (at 40 - 50 0C) to constant weight. The above procedure provided a final yield of Trilostane (2) of 9.5 g (95%). REFERENCES

1. Neumann H. C, Potts G. 0., Ryan W. T. and Stoner F. W., "Steroidal Heterocycles. XIII. 4α,5-Epoxy-5α-androst-2-eno[2,3-d]isoxazoles and Related Cmpounds", J. Med. Chem., 1970, 13, 948-951.