Methods for preparing 7alpha-hydroxy-dehydroepiandrosterone

This application is a 371 of PCT/FR02/00477 filed Feb. 07, 2002.

The invention relates to new processes for preparing 7alpha-hydroxy-dehydroepiandrosterone of formula (1):

7alpha-Hydroxy-dehydroepiandrosterone (7α-OH-DHEA) is a derivative well known in the scientific literature; the following documents may be cited in particular:

FR-A1-2 771 105; WO-A1-94/03176 and WO-A1-92/03925.

Each of the aforementioned documents reports a synthesis process for 7α-OH-DHEA. FR-A1-2 771 105 describes a process for preparing 7α-OH-DHEA in one step from dehydroepiandrosterone (DHEA), using a fungus: Fusarium moniliforme . This bioconversion process allows the direct transformation of DHEA into its 7-alpha-hydroxylated derivative with a yield of 79%.

This process allows the preparation of 7α-hydroxy-DHEA in a single step, but uses Fusarium moniliforme , which is a microscopic phytopathogenic fungus. This fungus attacks maize and sorghum (see in particular a document published on the internet at the following site: http://pested.unl.edu/catmans/fum.skp/fumchp8.htm). In addition, Fusarium moniliforme secretes oncogenic toxins which may prove highly dangerous to humans (see in particular a document published on the internet at the following site: http://www.hsafety.unc.edu/Manuals/LabManual/app7a.htm).

WO-A1-94/03176 and WO-A1-92/03925 describe a 4-step chemical process for preparing 7α-OH-DHEA from 3-O-acetyl-DHEA.

The first step of this process makes it possible to obtain 3-O-acetyl-7-bromo-DHEA from 3-O-acetyl-DHEA (commercial starting product) by treatment with a brominating agent. The mixture of 3-O-acetyl-7-bromo-DHEA isomers obtained is unstable and must be used quickly in the remainder of the process.

In a second step the racemic mixture of 7α-bromo and 7β-bromo isomers is equilibrated to give the 7α-bromo isomer (the more thermodynamically stable isomer) in majority form.

Following equilibration, the 3-O-acetyl-7-bromo-DHEA, treated with a mixture of glacial acetic acid and silver acetate, makes it possible to obtain 3-O-acetyl-7α-O-acetyl-DHEA.

3-O-Acetyl-7α-O-acetyl-DHEA, treated with sodium carbonate (Na 2 CO 3 ) in a mixture consisting of water and methanol, gives 7α-OH-DHEA, which is subsequently purified by crystallization.

This 4-step synthesis process has the prime disadvantage of necessitating a non-stereoselective bromination step (second step), which leads to unstable brominated intermediates. Furthermore, the process necessitates an equilibration step, permitting the thermodynamic isomer (7α-bromo-3-O-acetyl-DHEA) to be obtained predominantly. The equilibration step as described in WO-A1-94/03176 and WO-A1-92/03925 is ambiguous, since the relative proportion of the isomers 7α- and 7β-bromo-3-O-acetyl-DHEA obtained after equilibration is not specified.

It should also be noted that the process described does not report the yield obtained and does not specify the optical purity of the resultant 7α-OH-DHEA, this compound being documented solely by its melting point following recrystallization (192-193° C.).

Furthermore, the deacetalization conditions which are reported in the prior art do not allow satisfactory production of 7α-OH-DHEA from 3-O-acetyl-7α-OH-DHEA.

These conditions consist

either in a conventional method of saponification, using sodium hydroxide in an aqueous medium,

or in a method described in WO-A1-94/03176 and WO-A1-92/03925, which involves a reaction of double deacetalization of 3-O-acetyl-7-O-acetyl-DHEA by treatment with sodium carbonate in a mixture consisting of water and methanol.

These methods, applied to the reaction of deacetalization of 3-O-acetyl-7-O-acetyl-DHEA or of 3-O-acetyl-7α-OH-DHEA, lead to mixtures of products in which 7α-OH-DHEA occurs predominantly. This is because the reaction conditions used are not adapted and lead to the formation of decomposition products including in particular those obtained from the dehydration of the alcohol in position 7 of 3-O-acetyl-7α-OH-DHEA. There is therefore understandable interest in implementing processes which allow the preparation of 7α-OH-DHEA simply and rapidly, with industrially acceptable yields, with a diastereoselectivity which does not require a step of equilibration or of removal of the minority diastereoisomer (7β-OH), the processes having the advantage, moreover, of not involving phytopathogenic and/or oncogenic microorganisms.

Furthermore, the processes according to the present invention also have the advantage of including a deacetalization reaction which allows 7α-OH-DHEA to be obtained from 3-O-acetyl-7α-OH-DHEA under satisfactory conditions without the formation of decomposition products.

This is the aim of the present invention, whose objects include providing novel synthesis processes for DHEA.

The present invention provides a process for preparing 7α-OH-DHEA of formula (1) from 3-O-acetyl-7α-OH-DHEA of formula (4) according to a step (iii) consisting in a transesterification reaction using an alkali metal alkoxide in the corresponding alcohol.

The transesterification reaction which is used in the process of the invention consists essentially in

a) dissolving 3-O-acetyl-7α-OH-DHEA in an alcoholic solvent selected from ethanol, methanol and tert-butanol, preferably methanol; b) adding between 1 and 3 equivalents, preferably between 1.5 and 3 equivalents, advantageously 1 equivalent of an alkali metal alkoxide, the alkoxide being selected preferably from the ethoxide, the tert-butoxide and the methoxide, advantageously the methoxide, and the alkali metal being selected preferably from lithium, sodium and potassium, advantageously sodium; c) stirring the reaction mixture for a period of between 0.5 and 12 h, preferably between 1 h and 5 h, advantageously for 2 h; d) evaporating the reaction solvent and diluting the mixture with water before extracting it with a solvent selected preferably from dichloromethane, ethyl acetate, chloroform and diethyl ether, advantageously dichloromethane; e) drying the organic phase and then evaporating it to dryness; f) optionally purifying the residue, for example by chromatography.

Advantageously the invention relates to a process for preparing 7α-OH-DHEA of formula (1) from 3-O-acetyl-7-oxo-DHEA of formula (3) comprising step (iii) as defined above, characterized in that step (iii) is preceded by a step (ii) comprising a regioselective and diastereoselective reduction of 3-O-acetyl-7-oxo-DHEA using L-Selectride® as reducing agent.

According to one preferred embodiment of the invention, the reduction of the enone of 3-O-acetyl-7-oxo-DHEA of formula (3) can be accomplished regio-selectively and diastereoselectively, without prior protection of the carbonyl in position 17, by using as reducing agent L-Selectride® or lithium tri-sec-butyl-borohydride (LiB[CH(CH 3 )C 2 H 5 ] 3 H) in accordance with a method described in Steroids , 1998, 63, 454-458, which has been modified.

This method consists essentially in

a) preparing a solution of 3-O-acetyl-7-oxo-DHEA in an anhydrous organic polar solvent selected from tetrahydrofuran (THF), benzene and diethyl ether, preferably THF; b) cooling the solution to a temperature of between −90 and −10° C., preferably between −80 and −60° C., advantageously to −78° C.; c) adding under an inert atmosphere between 0.9 and 1.1 equivalents of L-Selectride®, preferably between 0.95 and 1.05 and, advantageously, 1 equivalent; d) stirring the solution at a temperature of between −90 and −10° C., preferably between −80 and −60° C., advantageously at −78° C. for a period of between 1 and 10 h, preferably for 5 h; e) taking the temperature of the reaction mixture to between −10 and 10° C., preferably to 0° C.; f) adding aqueous sodium hydroxide (NaOH) solution, preferably at a concentration of between 1 and 10 M, advantageously 6 M, followed by aqueous hydrogen peroxide (H 2 O 2 ) solution, preferably at 30 to 50%, advantageously at 30%, and stirring at ambient temperature; g) diluting the reaction mixture in an organic polar solvent selected from diethyl ether, ethyl acetate, chloroform and dichloromethane, preferably diethyl ether; h) washing the mixture a number of times by aqueous acidic washing, preferably with at least one aqueous 5% citric acid solution, then washing with at least one aqueous basic solution, preferably with saturated potassium bicarbonate (KHCO 3 ) solution, then washing with at least one aqueous neutral solution; i) drying the organic phase and then evaporating it to dryness; j) optionally purifying the residue, for example by chromatography.

Advantageously the invention relates to a process for preparing 7α-OH-DHEA of formula (1) from 3-O-acetyl-DHEA of formula (2) which comprises steps (iii) and (ii) as described above, characterized in that step (ii) is preceded by a step (i) consisting in a reaction of oxidation in the allyl position of 3-O-acetyl-DHEA.

FIG. 1 illustrates this 3-step synthesis process for 7α-OH-DHEA in accordance with the invention.

According to the invention a first preferred embodiment of the oxidation reaction in the allyl position of 3-O-acetyl-DHEA of formula (2) is carried out according to a method described in Tetrahedron Letters , 1997, 38, 119-122, which has been modified. This document describes a method of allylic oxidation by a peroxide in the presence of a copper salt catalyst on steroid frameworks.

This method consists essentially in

a) dissolving 3-O-acetyl-DHEA in an organic solvent or a mixture of organic solvents, selected preferably from acetonitrile, cyclohexane, benzene, methanol, ethanol and tert-butanol, advantageously acetonitrile; b) adding under an inert atmosphere between 0.005 and 0.1 equivalent of copper, preferably 0.01 equivalent, preferably in the form of a copper salt, selected for example from copper iodide (CuI), copper bromide (CuBr), copper chloride (CuCl) and copper(II) chloride (CuCl 2 ), advantageously CuI; c) cooling the reaction mixture at a temperature of between −10 and 10° C., advantageously between 5 and 10° C.; d) adding dropwise between 5 and 10 equivalents, preferably 6 equivalents, of organic or inorganic peroxide, preferably organic peroxide, selected for example from alkyl hydroperoxide, advantageously t-butyl hydroperoxide (t-BuOOH); e) allowing the temperature of the reaction mixture to rise to ambient temperature and stirring it for a period of between 0.5 and 4 h, preferably 2 h; f) heating the mixture at a temperature of between 40 and 80° C., preferably between 50 and 70° C., advantageously at 50° C., for a period of between 4 and 72 h, preferably between 16 and 24 h, advantageously for 20 h; g) cooling the reaction mixture and adding it to an aqueous alkaline solution, preferably 10% sodium bicarbonate (NaHCO 3 ) solution; h) extracting the aqueous phase with an organic solvent, selected for example from diethyl ether, ethyl acetate, chloroform and dichloromethane, preferably diethyl ether, then washing the organic phases with an aqueous alkaline solution, preferably saturated aqueous NaHCO 3 solution, and optionally with saturated aqueous sodium chloride (NaCl) solution; i) drying the organic phases and evaporating them to dryness under vacuum; j) optionally purifying the residue in accordance with customary methods.

According to the invention a second preferred embodiment of the oxidation reaction in the allyl position of 3-O-acetyl-DHEA of formula (2) is carried out according to a method described in WO 92/03925 and WO 94/03176, which has been modified. These documents describe a method of allyl oxidation of 3-O-acetyl-DHEA by a mixture of acid anhydride, acetic acid and sodium acetate in the presence of chromium trioxide (CrO 3 ).

This method consists essentially in

a) preparing a mixture consisting of 3-O-acetyl-DHEA; acid anhydride, in an amount of preferably between 3 and 20 equivalents, advantageously 11 equivalents; acetic acid, in an amount of preferably between 20 and 120 equivalents, advantageously 66 equivalents; and sodium acetate, in an amount of preferably between 1 and 5 equivalents, advantageously 3 equivalents; b) subjecting the reaction mixture to stirring and heating it to a temperature of between 40 and 60° C., preferably between 56 and 58° C.; c) adding between 1 and 10 equivalents of an oxide, preferably between 2 and 6 equivalents, advantageously 3.5 equivalents, the oxide being selectable from an oxide of chromium, of manganese, of selenium and of magnesium, and being preferably an oxide of chromium, advantageously chromium trioxide (CrO 3 ); d) keeping the reaction mixture under stirring for a period of between 0.5 and 4 h, preferably for 1 h, at a temperature of between 40 and 60° C., preferably between 56 and 58° C.; e) cooling the reaction mixture and adding it, with stirring, to a solution consisting of water and ice; f) filtering off the resulting precipitate and then washing it a number of times with water; g) drying the residue and optionally purifying it in accordance with customary methods; preferably the product is recrystallized.

According to the invention a third preferred embodiment of the oxidation reaction in the allyl position of 3-O-acetyl-DHEA of formula (2) is carried out by a method described in WO 98/50409, which has been modified. This document describes a method of allyl oxidation using an alkali metal periodate and an alkyl hydroperoxide.

This method consists essentially in

a) dissolving 3-O-acetyl-DHEA of formula (2) in an organic solvent or an organic solvent mixture, selected preferably from acetone, heptane, acetonitrile, tert-butanol, pyridine, petroleum ether, hexane, isooctane and cyclohexane, advantageously in a mixture consisting of acetone and heptane, very preferably in a 1/1 ratio; b) adding between 10 and 15 equivalents of alkyl hydroperoxide, advantageously t-butyl hydroperoxide, preferably at 70% or 80% in water; c) stirring the mixture at ambient temperature and adding between 1 and 5 equivalents, preferably 3 equivalents, of alkali metal periodate, advantageously sodium periodate (NaIO 4 ); d) adding water in an amount sufficient to allow total solubilization of the periodate in the reaction mixture, advantageously between 10% and 50% by volume, preferably between 15% and 40% by volume, very preferably between 20% and 30% by volume; e) adding between 1 and 5 equivalents, preferably 3 equivalents, of an organic base, preferably pyridine, or a weak inorganic base, selected preferably from alkali metal carbonates and bicarbonates, advantageously sodium bicarbonate; f) stirring the solution for a period of between 8 and 48 h, preferably for 14 h, at ambient temperature; g) diluting the reaction mixture with ethyl acetate, dichloromethane, chloroform or diethyl ether, preferably ethyl acetate; h) washing the mixture a number of times with aqueous sodium sulphate solution and then with water; i) drying the organic phase and then evaporating it to dryness; j) optionally purifying the residue in accordance with customary methods, preferably by recrystallization.

According to the invention an inert atmosphere means argon or nitrogen and ambient temperature means a temperature between 15 and 25° C. The purification methods which can optionally be employed at the end of each of the steps of the processes in accordance with the invention are realized according to conventional methods used in organic synthesis.

As mentioned above, the reaction of reduction of 3-O-acetyl-DHEA by L-Selectride® is diastereo-selective: the diastereoisomer obtained selectively is 3-O-acetyl-7α-OH-DHEA. On the other hand the reaction of reduction of 3-O-acetyl-DHEA by L-Selectride® may give rise to a not insignificant amount of product reduced in positions 7 and 17. This secondary product can be easily removed, for example by chromatography, at this stage or after the transesterification reaction of step (iii).

However, in order to prevent the formation of the product reduced in positions 7 and 17 in step (ii), a process for preparing 7α-OH-DHEA in accordance with the present invention may further comprise additional steps of protection and then deprotection of the carbonyl in position 17 of the reacting species.

Thus the invention relates advantageously to a process for preparing 7alpha-hydroxy-dehydroepiandrosterone of formula (1) from 3-O-acetyl-dehydroepiandrosterone, comprising steps (i), (ii) and (iii) as described above, characterized in that it further comprises the following additional steps:

(iv) a reaction of protection of the carbonyl in position 17 of 3-O-acetyl-DHEA before step (i); (v) a reaction of deprotection of the carbonyl in position 17 of the derivative obtained after step (ii), the said reaction of deprotection taking place before step (iii).

The protection of the carbonyl in position 17 of 3-O-acetyl-DHEA can be carried out in accordance with methods conventionally used in organic synthesis and well known to the person skilled in the art, such as, for example, those described in Protective Groups in Organic Synthesis , T. W. Greene, P. G. M. Wuts (Wiley Interscience).

According to the invention the carbonyl in position 17 of 3-O-acetyl-DHEA can be protected in cyclic or non-cyclic acetal form, in cyclic or non-cyclic dithioacetal form, in monothioacetal form, in O-substituted cyanohydrin form, in substituted hydrazone form, or in imine, enamine, imidazolidine or benzothiazole form. Advantageously the carbonyl in position 17 of 3-O-acetyl-DHEA can be protected in cyclic or non-cyclic acetal, cyclic or non-cyclic dithioacetal, imine, enamine or imidazolidine form.

According to the invention a first preferred embodiment of the protection of the carbonyl in position 17 of 3-O-acetyl-DHEA according to step (iv) can be carried out by protecting the carbonyl in position 17 in cyclic acetal form in accordance with a method described in Synth. Comm . 1995, 25, 395-404, which has been modified.

This method consists essentially in

a) preparing a solution consisting of 3-O-acetyl-DHEA of formula (2), a diol, preferably ethylene glycol, and an excess of desiccant, selected preferably from molecular sieve and ethyl orthoformate, preferably ethyl orthoformate, advantageously in a respective ratio of 1 mmol/3 ml/2 ml;

b) adding a catalytic amount of proton, the source thereof being advantageously para-toluenesulphonic acid or ion exchange resins;

c) stirring the mixture at a temperature of between 30 and 60° C., preferably at 40° C., for a period of between 1 and 8 h, preferably 3 h;

d) bringing the temperature of the reaction mixture to ambient temperature;

e) adding aqueous NaHCO 3 solution;

f) extracting the mixture with a solvent selected from dichloromethane, chloroform, ethyl acetate and diethyl ether, preferably with dichloromethane;

g) washing the organic phase, drying it and then evaporating it to dryness;

h) optionally purifying the residue in accordance with customary methods, preferably by chromatography on silica gel.

According to the invention a second preferred embodiment of the protection of the carbonyl in position 17 of 3-O-acetyl-DHEA according to step (iv) may be carried out by protecting the carbonyl in position 17 in cyclic acetal form in accordance with an azeotropic distillation method described in Synth. Comm . 1995, 25, 395-404, which has been modified.

This method consists essentially in

a) bringing to reflux a mixture consisting of 3-O-acetyl-DHEA of formula (2), ethylene glycol and para-toluenesulphonic acid (p-TsOH) in solution in benzene, preferably in a respective ratio of 1 mmol/5 ml/0.1 mmol/20 ml, in an assembly allowing azeotropic distillation to be conducted, preferably equipped with a Dean-Stark apparatus;

b) heating the mixture at a temperature of between 80 and 100° C., preferably at reflux of the benzene, for a period of between 4 and 48 h, preferably for 24 h;

c) allowing the temperature of the reaction mixture to return to ambient temperature;

d) drying the aqueous phase and then evaporating it to dryness;

e) optionally purifying the residue in accordance with customary methods, preferably by recrystallization.

The methods of deprotection of the carbonyl in position 17 of 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 can be carried out in accordance with methods conventionally used in organic synthesis and well known to the person skilled in the art, such as, for example, those described in Protective Groups in Organic Synthesis , T. W. Greene, P. G. M. Wuts (Wiley Interscience).

Thus according to the invention a first preferred embodiment of the deprotection of the carbonyl in position 17 of 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 in acetal form, preferably in cyclic acetal form, according to step (v) can be carried out in accordance with a method described in Steroids 1996, 61, 453-460, which has been modified.

This method consists essentially in

a) dissolving 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 in acetal form in a mixture comprising water and a water-miscible solvent, preferably acetone, preferably in a 1/1 ratio by volume;

b) adding para-toluenesulphonic acid (p-TsOH), preferably 1 equivalent;

c) stirring the solution for a period of between 4 and 48 h, preferably 24 h, at ambient temperature;

d) evaporating the water-miscible solvent under vacuum;

e) diluting the reaction mixture with an organic solvent, selected preferably from ethyl acetate, dichloromethane, chloroform and diethyl ether, advantageously ethyl acetate, then washing it a number of times with water;

f) drying the organic phase and then evaporating it to dryness;

g) optionally purifying the residue in accordance with customary methods, preferably by recrystallization.

According to the invention a second preferred embodiment of the deprotection of the carbonyl in position 17 of the 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 in acetal form, preferably cyclic acetal form, according to step (v) can be carried out in accordance with a method described in Steroids 1996, 61, 453-460, which has been modified.

Thus according to this other preferred embodiment of the invention the carbonyl in position 17 of the 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 in acetal form, preferably in cyclic acetal form, is deprotected by a method described in Synth. Comm . 1995, 25, 395-404, which has been modified.

This method consists essentially in

a) preparing a solution consisting of 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 in acetal form, preferably in cyclic acetal form, corresponding advantageously to 1,3-dioxolane or to 1,3-dioxane, in an organic solvent selected from benzene, chloroform and dichloromethane, preferably chloroform;

b) adding copper sulphate adsorbed on silica in an amount of between 2 and 4 g, preferably 2.5 g, per mmol of 3-O-acetyl-7α-hydroxy-DHEA protected in position 17 in acetal form;

c) stirring the reaction mixture for a period of between 1 to 48 h, preferably between 2 and 20 h, advantageously 6 h at a temperature of between 20 and 80° C., preferably between 50 and 70° C., advantageously at 60° C.;

d) filtering off the silica and concentrating the mixture to dryness;

e) optionally purifying the residue in accordance with customary methods, preferably by recrystallization.

The invention is illustrated in more detail in the following examples, which describe the various steps of the process in accordance with the invention.