COMPOUNDS

Field of the Invention

The invention relates to a process for obtaining Estetrol and 3-OH protected derivatives thereof, as well as to said 3-OH protected derivatives and intermediate products useful in the process.

Background of the Invention

Estrogenic substances are commonly used in methods of Hormone Replacement

Therapy (HRT) and methods of female contraception. These estrogenic substances can be divided in natural estrogens and synthetic estrogens. Examples of natural estrogens that have found pharmaceutical application include estradiol, estrone, estriol and conjugated equine estrogens. Examples of synthetic estrogens, which offer the advantage of high oral bioavailability include ethinyl estradiol and mestranol.

Estetrol [estra-1 ,3,5(10)-trien-3,15a,16a,173-tetraol; CAS Nr. 15183-37-6] is a biogenic estrogen that is endogeneously produced by the fetal liver during human pregnancy. In this description the lUPAC-recommended ring lettering and atom numbering for steroids and steroid derivatives as depicted below, are applied.

estetrol

Estetrol has been found effective as an estrogenic substance for use in HRT (EP 1 390 040 B1 , EP 1 446 128 B1 ), contraception (EP 1 390 041 B1 , EP 1 390 042 B1 ), therapy of auto-immune diseases (EP 1 51 1 496 B1 ), prevention and therapy of breast and colon tumors (EP 1 526 856 B1 ), enhancement of libido (EP 1 390 039 B1 ), treatment of infertility (EP 2 1 14 412 B1 ), treatment of acute vascular disorder (EP 1 971 344 B1 ), skin care and wound healing (EP 1 51 1 498 A1 ).

The synthesis of Estetrol on a laboratory scale is for example disclosed in Fishman et al., J. Org. Chem. 1968, 33, 3133 - 3135, wherein Estetrol is synthesized from estrone derivative III as shown in Scheme 1 (numbering according to Fishman et al).

Scheme 1

According to Fishman et al., oxidation of the allylic diacetate Vlb with Os0 ₄ produced as the desired product lb together to a small amount of the isomeric 15β,16β- diol. The authors did not quantify the isomeric mixture. The yield of the dihydroxylation is 47% and the overall yield of the 3-step process shown in Scheme 1 is, starting from estrone derivative III, about 7%.

Another synthesis of Estetrol wherein estrone is the starting material is disclosed in Nambara et al., Steroids 1976, 27, 1 1 1 - 121 . This synthesis is shown in Scheme 2 (numbering according to Nambara et al.). The carbonyl group of estrone I is first protected by treatment with ethylene glycol and pyridine hydrochloride followed by acetylation of the hydroxyl group at C3. The next sequence of steps involved a bromination/base catalyzed dehydrobromination resulting into the formation of 17,17- ethylenedioxyestra-1 ,3,5(10),15-tetraene-3-ol (compound IVa). This compound IVa was subsequently acetylated which produced 17,17-ethylenedioxyestra-1 ,3, 5(10), 15- tetraene-3-ol-3-acetate (compound IVb). In a next step, the dioxolane group of compound IVb was hydrolysed by using p-toluene sulfonic acid to compound Vb, followed subsequently by reduction of the carbonyl group at C17 (compound Vc), acetylation (compound Vd) and oxidation of the double bond of ring D thereby forming estra-1 ,3,5(10)-triene-3,15a,16a,173-tetraol-3,17-diacetate (compound Vlb). Neither experimental protocol nor details about the yield or selectivity of said oxidation of the double bond of ring D are provided in Nambara et al..

1. P_M¾ NBr.Bi

2. t-BuOK, DMSO

Vb IV»

1. Li Ml

2. At¾G,pyr

3. ( ¾

VII»

Scheme 2

Suzuki et al., Steroids 1995, 60, 277 - 284 also discloses the synthesis of Estetrol by using compound Vb of Nambara et al. as starting material. The carbonyl group at C17 of this compound was first reduced followed by acetylation yielding estra- 1 ,3,5(10),15-tetraene-3,17-diol-3, 17-diacetate (compound 2b). The latter was subjected to oxidation with Os0 ₄ which provided estra-1 ,3,5(10)-triene-3,15a,16a,173-tetraol-3, 17-diacetate (compound 3b) in 46% yield, along with the isomeric 15β, 1 θβ-diol as impurity (15α,16α / 15β,16β isomeric ratio = 74/26).

3b

According to Nambara et al. and Suzuki et al., the synthesis of Estetrol can be performed with a yield of approximately 8%, starting from estrone.

A process for the preparation of Estetrol that is suitable for the preparation of this compound on an industrial scale is disclosed in WO 2004/041839 A2. This process is shown in Scheme 4 (numbering according to WO 2004/041839), and comprises the following steps:

1 ) converting estrone (7) into 3-A-oxy-estra-1 ,3,5(10),15-tetraen-17-one (6), wherein A is a protecting group, this step involving in turn five sub-steps;

2) reduction of the 17-keto group of 3-A-oxy-estra-1 ,3,5(10),15-tetraen-17-one (6) to 3-A-oxy-estra-1 ,3,5(10),15-tetraen-173-ol (5);

3) protection of the 17-OH group of 3-A-oxy-estra-1 ,3,5(10),15-tetraen-173-ol (5) to 3-A-oxy-17-C-oxy-estra-1 ,3,5(10),15-tetraene (4), wherein C is a protecting group;

4) oxidizing the carbon-carbon double bond of ring D of 3-A-oxy-17-C-oxy- estra-1 ,3,5(10),15-tetraene (4) to protected Estetrol (3); and

5) removing the protecting groups, wherein preferably protecting group A is removed first to form 17-OC protected Estetrol (2) and subsequently protecting group C is removed to form Estetrol (1 );

wherein the protecting group A is selected from an Ci-C ₅ alkyl group or a C ₇ Ci2 benzylic group and the protecting group C is selected from monofunctional aliphatic hydroxyl protecting groups.

1

cstctiol

Scheme 4

With the method as disclosed in WO 2004/041839 and shown in Scheme 4 above, Estetrol is obtained in an overall yield of 10.8%, starting from estrone. Specifically, the yield of the cis-dihydroxylation step as described in the example 9 is 43% after three crystallizations in order to purify the product from the 15β,1 θβ-diol isomer. Although the process disclosed in WO 2004/041839 is suitable for an industrial scale preparation of Estetrol, the high number of synthetic steps and the isolation and purification of each intermediate product results in a loss of yield, thereby reducing the overall yield of Estetrol. Furthermore, the conversion of 7 into 6 involves a halogenation and a dehalogenation step, typically a bromination and a debromination step. In particular during said halogenation and dehalogenation reactions, various side products are produced.

Another process for the preparation of Estetrol on an industrial scale is disclosed in EP 2383279 A1 . This process, depicted in Scheme 5 below, comprises the following steps:

(l )conversion of estrone II into 17-B-oxy-3-A-oxy-estra-1 ,3,5(10),16-tetraene III, wherein A is a protecting group and B is -Si(R ² ) ₃ ; (2) conversion of 17-B-oxy-3-A-oxy-estra-1 ,3,5(10),16-tetraene III into 3-A-oxy- estra-1 ,3,5(10),15-tetraen-17-one IV, wherein A is a protecting group;

(3) reduction of the 17-keto group of 3-A-oxy-estra-1 ,3,5(10),15-tetraen-17-one IV to form 3-A-oxy-estra-1 ,3,5(10),15-tetraen-173-ol V, wherein A is a protecting group;

(4) protection of the 17-OH group of 3-A-oxy-estra-1 ,3,5(10),15-tetraen-173-ol V to form 3-A-oxy-17-C-oxy-estra-1 ,3,5(10),15-tetraene (VI), wherein A and C are protecting groups;

(5) oxidation of the carbon-carbon double bond of ring D of 3-A-oxy-17-C-oxy- estra-1 ,3,5(10),15-tetraene (VI) to form protected Estetrol VII, wherein A and C are protecting groups; and

(6) removal of protecting groups A and C to form Estetrol I;

wherein:

A is a protecting group selected from the group consisting of a Ci-C ₅ alkyl group, a C7-C12 benzylic group and a-Si(R ¹ ) ₃ group, wherein R ¹ is independently selected from the group consisting of a C C ₆ alkyl group and a C ₆ -Ci ₂ aryl group;

B is -Si(R ² ) ₃ , wherein R ² is independently selected from the group consisting of a Ci-C ₆ alkyl group and a C ₆ -Ci ₂ aryl group; and

C is a protecting group selected from the group consisting of monofunctional aliphatic hydroxyl protecting groups.

III IV

¥1

Scheme 5

The yield reported for step 5, oxidation of the carbon-carbon double bond from the intermediate product VI to form protected Estetrol VII, is 43% after purifications, with a purity of 98.7%. If a Palladium catalyst is used, the cost of the process increases to a large extent.

All the processes disclosed in the state of the art comprise a high number of synthetic steps, due to inter alia the protection/deprotection of the hydroxyl groups at the 3- and 17-positions, which affect the overall yield in which Estetrol is obtained. Further, the oxidation of the carbon-carbon double bond of ring D typically proceeds with a poor stereoselectivity, obtaining large amounts of the undesired isomeric 15β,163-diol. Accordingly, the preparations of Estetrol known so far cannot offer a good yield and high purity, required to an effective industrial production.

In view of the above, it is still necessary to provide an alternative process for obtaining Estetrol on an industrial scale, which allows the production of this compound in a high yield, and at the same time, minimizes the impurities associated. Summary of the Invention

The invention faces the problem of providing an efficient process for the preparation of Estetrol. The inventors have surprisingly found a process for obtaining Estetrol in which the oxidation of the double bond of ring D proceeds over a starting material having the β-hydroxyl group at C17 free, avoiding therefore the need of using the corresponding protecting groups. Unexpectedly, not only the free hydroxyl group at C17 is not oxidized during the process but also the oxidation of the carbon-carbon double bond of ring D shows a high stereoselectivity to afford the desired 15a,16a-diol.

Thus, in one aspect the present invention refers to a process for the preparation of a compound of formula (I)

(I)

or a salt or solvate thereof,

wherein R represents H or an hydroxyl protecting group (HPG);

the process comprising the oxidation of the carbon-carbon double bond of ring D of a compound of formula (II)

(ii)

or a salt or solvate thereof, wherein R is as defined previously.

If R is H, the compound of formula (I) is Estetrol. If R is not H, the process preferably further comprises the deprotection of the hydroxyl group to give Estetrol.

In a preferred embodiment, the compound of formula (II) or a salt or solvate thereof is prepared by a process comprising the reduction of the keto group at C17 of a compound of formula (III)

(in)

or a salt or solvate thereof, wherein R is as defined previously.

In another aspect the present invention refers to a compound of formula (I)

(I)

or a salt or solvate thereof,

wherein R represents an hydroxyl protecting group selected from:

- silyl ethers [-Si(R _x )(R _y )(R _z )], where R _x , R _y and R _z can be independently selected from alkyl, cycloalkyi, aryl, alkoxy and halogen;

- ethers [-R], where R can be selected from from alkyl, cycloalkyi, aryl and arylalkyi, with the proviso that R is not butanamide-4-oxy, butanoic acid 4-oxy, ethyl butanoate-4-oxy and β-D-Glucopyranosiduronic acid;

- alkoxy and aryloxy alkyl ethers such as alkoxy and aryloxy methyl ether [-CH ₂ - OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH ₂ -ORw], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyi;

- esters [-COR _w ], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyi, with the proviso that CORw is not acetyl, 4-(aminosulfonyl)benzoyl or 3- (aminosulfonyl)benzoyl;

- amides [-CONR _w R _v ], where R _w and R _v can be independently selected from alkyl, cycloalkyi, aryl and arylalkyi; and

- carbonates [-COOR _w ], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyi.

In another aspect the present invention refers to a compound of formula (II)

(ii)

or a salt or solvate thereof,

wherein R represents an hydroxyl protecting group selected from:

- ethers [-R], where R can be selected from alkyl, cycloalkyi, aryl and arylalkyi, with the proviso that R is not C1 -C5 alkyl; - alkoxy and aryloxy alkyl ethers such as alkoxy and aryloxy methyl ether [-CH ₂ - OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH2-OR _w ], where R _w can be selected from alkyl, cycloalkyl, aryl and arylalkyl;

- esters [-COR _w ], where R _w can be selected from alkyl, cycloalkyl, aryl and arylalkyl, with the proviso that COR _w is not acetyl or C7-C12 benzoyl;

- amides [-CONR _w R _v ], where R _w and R _v can be independently selected from alkyl, cycloalkyl, aryl and arylalkyl; and

- carbonates [-COOR _w ], where R _w can be selected from alkyl, cycloalkyl, aryl and arylalkyl.

These aspects and preferred embodiments thereof are additionally also defined hereinafter in the detailed description, as well as in the claims.

Detailed Description of the Invention

Definitions

In the context of the present invention, the following terms have the meaning detailed below.

As used herein, the term "alkyl" refers to a linear or branched alkane derivative containing from 1 to 6 ("CrC ₆ alkyl"), preferably from 1 to 3 ("CrC ₃ alkyl"), carbon atoms and which is bound to the rest of the molecule through a single bond. Illustrative examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, pentyl, hexyl, etc.

The term "alkoxy" refers to a radical of the formula -OR where R is an alkyl radical as defined above having one or more (e.g., 1 , 2, 3 or 4) oxygen linkages and from 1 to 6 carbon atoms or preferably 1 to 3 carbon atoms, e. g., methoxy, ethoxy, propoxy, etc. The term "aryloxy" refers to a radical of formula -OR wherein R is an aryl radical as defined below, e.g., -O-phenyl, -O-p-tolyl, -O-m-tolyl, -O-o-tolyl or -O- naphtyl.

The term "aryl" refers to an aromatic group having between 6 and 18 ("C ₆ -Ci ₈ aryl"), preferably between 6 and 10 ("C ₆ -Ci ₀ aryl"), more preferably 6 or 10 carbon atoms, comprising 1 , 2 or 3 aromatic nuclei bound through a carbon-carbon bond or fused to one another. Illustrative examples of aryl groups include phenyl, naphthyl, biphenyl, indenyl, phenanthryl, etc.

The term "arylalkyl" refers to an alkyl group as defined above substituted with an aryl group as defined above, such as (C6-Ci8)aryl(Ci-C ₆ )alkyl, (C6-Cio)aryl(Ci-C ₆ )alkyl and (C ₆ -Cio)aryl(Ci-C ₃ )alkyl. Examples of such groups include benzyl, phenylethyl, phenylpropyl, naphthylmethyl, etc.

The term "cycloalkyl" refers to a radical derived from cycloalkane containing from 3 to 7 ("C3-C7 cycloalkyl"), preferably from 3 to 6 ("C3-C6 cycloalkyl") carbon atoms. Illustrative examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

The term "halogen" refers to bromine, chlorine, iodine or fluorine.

"Heterocyclyl" refers to a stable cyclic radical of 3 to 10 members, preferably a cycle of 5 or 6 members consisting of carbon atoms and from 1 to 5, preferably from 1 to 3, heteroatoms selected from nitrogen, oxygen and sulfur, and which may be completely or partially saturated or be aromatic ("heteroaryl"). In the present invention, the heterocyclyl can be a mono-, bi- or tricyclic system which may include fused ring systems. Illustrative examples of heterocyclyl groups include, for example, pyrrolidine, piperidine, piperazine, morpholine, tetrahydrofuran, benzimidazole, benzothiazole, furan, pyrrole, pyridine, pyrimidine, thiazole, thiophene, imidazole, indole, etc.

As understood in this technical area, there may be a certain degree of substitution in the aforementioned radicals. Therefore, there may be substitution in any of the groups of the present invention. The previous groups can be substituted in one or more available positions with one or more substituents. Said substituents include, for example and in non-limiting sense, alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, halogen, -CN, N0 ₂ , CF ₃ , -N(R _a )(R _b ), -OR _c , -SR _d , -C(0)R _e , -C(0)OR _f , -C(0)N(R _g )(R _h ), - OC(0)Ri; wherein R _a , R _b , R _c , Rd, R _e , R _f , R _g , R _h and R, are independently selected from hydrogen, alkyl, aryl, heterocyclyl, heteroaryl and trifluoromethyl.

The term "hydroxyl protecting group" (HPG) refers to a group blocking the OH function for subsequent reactions that can be removed under controlled conditions. Hydroxyl protecting groups are well known in the art. Illustrative examples of hydroxyl protecting groups have been described by Green TW et al. in "Protective Groups in Organic Synthesis", 3rd Edition (1999), Ed. John Wiley & Sons (ISBN 0-471 -16019-9). Virtually any hydroxyl protecting group can be used to put the invention into practice. Illustrative, non-limiting examples of HPGs include:

- silyl ethers [-Si(R _x )(R _y )(R _z )]. R _x , R _y and R _z can be independently selected from alkyl, cycloalkyl, aryl, alkoxy and halogen. Examples of silyl ethers include trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert- butyldiphenylsilyl ether, tri-isopropylsilyl ether, diethylisopropylsilyl ether, thexyldimethylsilyl ether, triphenylsilyl ether, di-tert-butylmethylsilyl ether, dimethylphenyl ether;

- ethers [-R]. R can be selected from alkyl, cycloalkyl, aryl and arylalkyl. Examples of ethers include methyl ether, tert-butyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzyl ether, trityl ether, allyl ether;

- alkoxy and aryloxy alkyl ethers such as alkoxy and aryloxy methyl ether [-CH ₂ - OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH ₂ -ORw]. R _w can be selected from alkyl, cycloalkyl, aryl and arylalkyl. Examples of alkoxy and aryloxy alkyl ethers include methoxymethyl ether, 2-methoxyethoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, 2-

(trimethylsilyl)ethoxymethyl ether, 1 -methoxyethyl ether, 1 -ethoxyethyl ether, 1 -n- propoxyethyl ether, 1 -isopropoxyethyl ether, 1 -n-butoxyethyl ether, 1 - isobutoxyethyl ether, 1 -sec-butoxyethyl ether, 1 -tert-butoxyethyl ether, 1 -ethoxy- n-propyl ether, methoxypropyl ether, ethoxypropyl ether, 1 -methoxy-1 -methylethyl ether, 1 -ethoxy-1 -methylethyl ether; tetrahydropyranyl and related ethers;

- esters [-COR _w ]. R _w can be selected from alkyl, cycloalkyl, aryl and arylalkyl.

Examples of esters include acetyl, benzoyl, pivaloyl, methoxyacetyl, chloroacetyl, levulinyl ester;

- amides [-CONR _w R _v ]. R _w and R _v can be independently selected from alkyl, cycloalkyl, aryl and arylalkyl; and

- carbonates [-COOR _w ]. R _w can be selected from alkyl, cycloalkyl, aryl and arylalkyl. Examples of carbonates include benzyl carbonate, p-nitrobenzyl carbonate, tert-butyl carbonate, 2,2,2-trichloroethyl carbonate, 2- (trimethylsilyl)ethyl carbonate, allyl carbonate.

The invention also provides "salts" of the compounds described in the present description. By way of illustration, said salts can be acid addition salts, base addition salts or metal salts, and can be synthesized from the parent compounds containing a basic or acid moiety by means of conventional chemical processes known by the persons skilled in the art. Such salts are generally prepared, for example, by reacting the free acid or base forms of said compounds with a stoichiometric amount of the suitable base or acid in water or in an organic solvent or in a mixture of the two. Nonaqueous media such as ether, ethyl acetate, ethanol, acetone, isopropanol or acetonitrile are generally preferred. Illustrative examples of said acid addition salts include inorganic acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc., organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, etc. Illustrative examples of base addition salts include inorganic base salts such as, for example, ammonium salts and organic base salts such as, for example, ethylenediamine, ethanolamine, Λ/,/V-dialkylenethanolamine, triethanolamine, glutamine, amino acid basic salts, etc. Illustrative examples of metal salts include, for example, sodium, potassium, calcium, magnesium, aluminum and lithium salts.

Likewise, the compounds described in the present description can be obtained both as free compounds or as solvates (e.g., hydrates, alcoholates, etc.), both forms being included within the scope of the present invention. The solvation methods are generally known in the state of the art.

The term "pharmaceutically acceptable" relates to molecular entities and compositions being physiologically tolerable and normally not causing an allergic reaction or similar adverse reaction, such as gastric discomfort, dizziness and the like, when they are administered to a human being. Preferably, as used in this description, the term "pharmaceutically acceptable" means approved by a governmental regulatory agency or listed in the US pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more particularly in humans.

For those persons skilled in the art, it will be evident that the scope of the present invention also includes salts which are not pharmaceutically acceptable as possible means for obtaining pharmaceutically acceptable salts.

Unless otherwise indicated, the compounds of the invention also include compounds which differ in the presence of one or more isotopically enriched atoms. By way of illustration, compounds having the structures defined herein, with the exception of the substitution of at least one hydrogen by a deuterium or tritium, or the substitution of at least one carbon by a carbon enriched in ¹³ C or ¹⁴ C, or at least one nitrogen by a nitrogen enriched in ¹⁵ N, are within the scope of this invention.

As used herein, the term "about" means a slight variation of the value specified, preferably within 10 percent of the value specified. Nevertheless, the term "about" can mean a higher tolerance of variation depending on for instance the experimental technique used. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. Further, to provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about". It is understood that, whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. In an aspect, the invention refers to a process for the preparation of a compound of formula (I)

(I)

or a salt or solvate thereof,

wherein R represents H or an hydroxyl protecting group (HPG);

the process comprising reacting a compound of formula (II)

(ii)

or a salt or solvate thereof, wherein R is as defined previously,

with an oxidizing agent. According to the state of the art (Warmerdam et al. Climateric (2008), 1 1 (Suppl

1 ) 59-63), it is essential for the cis-dihydroxylation reaction that the hydroxyl groups at position 3 and 17 are protected. To the best knowledge of the inventors, this is the first time that it is disclosed the oxidation over a starting material without hydroxyl protecting groups at C17 in order to prepare Estetrol. This allows reducing the number of synthetic steps, thus increasing the overall yield and simplifying the synthesis. Moreover, contrary to the teachings of the state of the art, which suggest that the protection of the hydroxyl at C17 increases the steric hindrance in the β-face, improving the formation of the isomeric α,α-diol, the process of the invention has been found to provide high stereoselectivities in favour of the desired α,α-isomer as well, typically≥ 90%.

The oxidation of the carbon-carbon double bond in ring D of the compound of formula (II) is carried out with an oxidizing agent providing selective cis-hydroxylation of the carbon-carbon double bond to render a compound of formula (I). Preferably, the oxidation reagent is Os0 ₄ or a source of osmium tetroxide such as potassium osmate(VI) dihydrate (K ₂ 0s0 ₄ -2H ₂ 0) or osmium(lll) chloride hydrate (OsCI ₃ -xH ₂ 0) which easily oxidise to osmium(VIII). More preferably Os0 ₄ (or a source of Os0 ₄ ) supported/immobilized such as osmium tetroxide supported on poly(4-vinyl-pyridine) (Os0 ₄ -PVP) (cf. G. Cainelli et al., Synthesis 1989, 45 - 47), AD-mix (alpha and beta) or Os Encat™. In a particular embodiment, the amount of Os0 ₄ supported on PVP is about 5%. AD mix-a is a commercially available mixture containing (DHQ) ₂ PHAL (hydroquinine 1 ,4-phthalazinediyl diether) 0.0016 mole, potassium carbonate, powder 0.4988 mole, potassium ferricyanide 0.4988 mole, and potassium osmate dihydrate 0.0007 mole. AD mix-β is a commercially available mixture containing (DHQD) ₂ PHAL (hydroquinidine 1 ,4-phthalazinediyl diether) 0.0016 mole, potassium carbonate, powder 0.4988 mole, potassium ferricyanide 0.4988 mole, and potassium osmate dihydrate 0.0007 mole. Os Encat™ is Os0 ₄ immobilized in a polyurea matrix; in particular Os EnCat™ 40 has the following properties: Os metal content 4.8 - 5.7 % w/w and Os0 ₄ loading 40 - 300 mmol/g (average 165 mmol/g).

In a preferred embodiment, an oxidation co-reagent or co-oxidant is added additionally, such as trimethylamine-N-oxide, triethylamine-N-oxide, dimethylbencilamine-N-oxide, N-methyl morpholine-N-oxide, TEMPO or hydrogen peroxide and derivatives, more preferably trimethylamine-N-oxide.

This reaction of cis-dihydroxylation is typically carried out in a suitable organic solvent, such as an ether, for example, an acyclic ether (e.g., diisopropylether, etc.) or a cyclic ether (e.g., tetrahydrofuran (THF), a dioxane, etc.), a halogenated solvent such as, for example, dichloromethane, etc., or in an aromatic solvent such as, for example, toluene, etc. Preferably, the solvent is THF. More preferably, Os0 -PVP (poly(4-vinyl- pyridine) and trimethylamine-N-oxide are used with THF as the solvent.

In a particular embodiment, the amount of the organic solvent may be a proportion between 3 and 20 mL per gram of the compound of formula (II), more preferably between 8 and 15 mL per gram of the compound of formula (II). Typically, the quantity of oxidation co-reagent may vary between 0.95 and 4 equivalents, more preferably between 1 .0 and 2.5 equivalents and the amount of the oxidation reagent may be used between 10% and 50% per gram of the compound of formula (II), more preferable between 15% and 25% per gram of the compound of formula (II).

While all the reagents may be added at room temperature, preferably under inert atmosphere, then the mixture is preferably heated such as at a temperature comprised between room temperature and 100°C. The reaction rate depends on the particular conditions including the temperature, where at a temperature comprised between 55°C and 60°C the reaction usually takes place in a time period of between 20-24 hours.

The compound of formula (I) obtained in the oxidation can be isolated by means of conventional techniques and/or, when R is not H, transformed into Estetrol. Thus, Estetrol can be prepared from a compound of formula (I) wherein R is a hydroxyl protecting group by conventional methods of deprotection known by persons skilled in the art (Green TW et al. in "Protective Groups in Organic Synthesis", 3rd Edition (1999), Ed. John Wiley & Sons (ISBN 0-471 -16019-9). The progress of the reaction of deprotection can be easily monitored by TLC.

For example, compounds of formula (I) wherein R represents an ester, a carbonate or an amide can be easily converted into Estetrol by hydrolysis in basic or acid media according to well-established procedures of the state of the art.

Compounds of formula (I) wherein R represents a silyl radical can be easily converted into Estetrol by the use of fluoride reagents such as fluoride salts or HF, acid media, oxidizing media, etc.

Compounds of formula (I) wherein R represents an ether can be easily converted into Estetrol through hydrolysis in acid media (for example, for methyl ethers), hydrogenation (for example, for benzyl ethers), oxidation (for example, for aryl ethers), etc. In general, the deprotection reaction of ethers of formula (I) to afford Estetrol provides quantitative yields.

In a preferred embodiment, the compound of formula (II) or a salt or solvate thereof is prepared by a process comprising reacting a compound of formula (III)

(III)

or a salt or solvate thereof, wherein R is as defined previously,

with a reducing agent.

This step can be carried out by means of any reduction reaction which allows the transformation of the keto group at C17 of a compound of formula (III) into a hydroxyl group to render a compound of formula (II). Thus, the reduction reaction can be carried out under conventional conditions known in the art. In a particular embodiment, the reaction is performed using a reducing agent selected from a metallic hydride such as sodium borohydride, sodium cyanoborohydride, potassium borohydride, potassium cyanobohydride and lithium aluminum hydride. In a preferred embodiment, the reducing agent is sodium borohydride, preferably in the presence of cerium trichloride (NaBH ₄ /CeCI ₃ ). More preferably, the reducing agent for use herein is NaBH ₄ in combination with CeCI ₃ hydrate, preferably cerium trichloride heptahydrate (CeCI ₃ ^■ 7H ₂ 0).

In a particular embodiment, the reduction reaction is carried out using between 1 and 10, preferably between 1 and 5, more preferably between 1 and 3 equivalents of the reducing agent per equivalent of compound of formula (III), or a salt or solvate thereof.

In a preferred embodiment, the reaction of the compound of formula (III) with the reducing agent is carried out in a mixture of a protic solvent, such as MeOH and THF. In particular, it is preferred to suspend the compound of formula (III) and the reducing agent in a mixture of a protic solvent, preferably MeOH and THF, at 0°C-5°C and to stir the mixture at said low temperature. A preferred volume ratio of MeOH to THF is 2: 1 to 5: 1 .

The preparation of the unsaturated estrone (III) is well known in the state of the art (see for instance, Cantrall et al., J. Org. Chem. 1964, 29, 214 - 217; Johnson et al., J. Am. Chem. Soc. 1957, 79, 2005 - 2009; Poirier et al., Tetrahedron 1991 , 47, 7751 - 7766; Nambara et al., Steroids 1976, 27, 1 1 1 - 121 ; Li et al.; Steroids 2010, 75, 859 - 869). According to a particular embodiment, the compound of formula (III) was prepared following the scheme depicted below in Scheme 6:

Unsaturated Estrone (III) (IV)

Scheme 6 The 3-OH group of the estrone is protected for instance by acylation, silylation, formation of an ether, etc. In the case of the acylation, the 3-OH group is preferably acylated using a reagent selected from benzoyl, benzyl or acetyl chloride in dichloromethane and triethylamine as base.

Then, the carbonyl group of intermediate product (VII) may be protected by treatment with ethylene glycol, triethylortoformiate and p-toluene sulfonic acid to render compound (VI) in practically quantitative yield.

Subsequently, an alpha-bromination may be carried out with pyridinium bromide in THF in presence of ethylene glycol to obtain the compound (V). In a particular embodiment, the amount of ethylene glycol may be from 5 to 25% with respect to the amount of THF.

The carbon-carbon double bond of ring D may be achieved with treatment of the compound (V) with i-BuOK in DMSO; under said conditions, if an ester is used as hydroxyl protecting group at C3 is unstable.

In a particular embodiment, the dehydrobromination reaction is carried out using between 1 and 10, more preferably between 2 and 6 equivalents of i-BuOK per equivalent of compound of formula (V) and the amount of DMSO may be between 5 and 20 mL per gram of the compound of formula (V), more preferably between 7 and 1 1 mL per gram of the compound of formula (V).

In a preferred embodiment the dehydrobromination reaction is carried out using between 2 and 6 equivalents of i-BuOK equivalent of compound of formula (V) and an amount of DMSO between 7 and 1 1 mL per gram of the compound of formula (V),

The compound of formula (IV) may be prepared from the compound of formula (VI) in a one-pot process, without isolating the intermediate compound (V). However, in a preferred variant of the invention, the compound of formula (V) is isolated.

The compound of formula (IV) obtained in the dehydrobromination reaction does not need further purifications (such as crystallizations or chromatographies) nor requires to be dried. Thus, the dioxolane group of compound (IV), without purification or drying, may be hydrolyzed by using p-toluene sulfonic acid to obtain unsaturated estrone (III) (R=H) in practically quantitative yield, which is then optionally protected (R= HPG) under conventional techniques.

The preparation of the unsaturated estrone (III) is not limited to the specific process shown in Scheme 6, but as the skilled person will appreciate, protection and deprotection of the hydroxyl group at position 3 and the keto group at position 17 can be performed at any stage of the synthesis. The most suitable stage for said protection and/or deprotection can be readily determined by those skilled in the art. In a particular embodiment, the hydroxyl group at position 3 is not protected during the process. A particular embodiment of the invention, wherein the 3-OH group is free during the w

Estetrol (I)

Scheme 7

In additional preferred embodiments, the preferences described above for the processes are combined. The present invention is also directed to such combinations of preferred conditions of the processes.

Compounds of formula (I) of the invention

In another aspect the present invention refers to a compound of formula (I) per se

(I)

or a salt or solvate thereof,

wherein R represents an hydroxyl protecting group selected from:

silyl ethers [-Si(R _x )(R _y )(R _z )], where R _x , R _y and R _z can be independently selected from alkyl, cycloalkyl, aryl, alkoxy and halogen; - ethers [-R], where R can be selected from from alkyl, cycloalkyi, aryl and arylalkyl, with the proviso that R is not butanamide-4-oxy, butanoic acid 4-oxy, ethyl butanoate-4-oxy and β-D-Glucopyranosiduronic acid;

- alkoxy and aryloxy alkyl ethers such as alkoxy and aryloxy methyl ether [-CH ₂ - OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH ₂ -ORw], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyl;

- esters [-COR _w ], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyl, with the proviso that CORw is not acetyl, 4-(aminosulfonyl)benzoyl or 3- (aminosulfonyl)benzoyl;

- amides [-CONR _w R _v ], where R _w and R _v can be independently selected from alkyl, cycloalkyi, aryl and arylalkyl; and

- carbonates [-COOR _w ], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyl.

In a particular embodiment in the compound of formula (I), R is a silyl ether [- Si(R _x )(Ry)(R _z )] selected from trimethylsilyl ether, triethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, tri-isopropylsilyl ether, diethylisopropylsilyl ether, thexyldimethylsilyl ether, triphenylsilyl ether, di-tert-butylmethylsilyl ether, dimethylphenyl ether.

In a particular embodiment in the compound of formula (I), R is an ether [-R] selected from alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, pentyl, hexyl; cycloalkyi such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; aryl such as phenyl, naphthyl, biphenyl, indenyl, phenanthryl; and arylalkyl such as benzyl, phenylethyl, phenylpropyl, naphthylmethyl.

In a particular embodiment in the compound of formula (I), R is a alkoxy or aryloxy alkyl ether such as alkoxy and aryloxy methyl ether [-CH ₂ -OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH ₂ -OR _w ] selected from methoxymethyl ether, 2-methoxyethoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, 2- (trimethylsilyl)ethoxymethyl ether, 1 -methoxyethyl ether, 1 -ethoxyethyl ether, 1 -n- propoxyethyl ether, 1 -isopropoxyethyl ether, 1 -n-butoxyethyl ether, 1 -isobutoxyethyl ether, 1 -sec-butoxyethyl ether, 1 -tert-butoxyethyl ether, 1 -ethoxy-n-propyl ether, methoxypropyl ether, ethoxypropyl ether, 1 -methoxy-1 -methylethyl ether, 1 -ethoxy-1 - methylethyl ether; tetrahydropyranyl and related ethers.

In a particular embodiment in the compound of formula (I), R is an ester [-COR _w ] selected from acetyl, benzoyl, pivaloyl, methoxyacetyl, chloroacetyl, levulinyl.

In a particular embodiment in the compound of formula (I), R is an amide [-CONR _w R _v ] such as dimethylamide.

In a particular embodiment in the compound of formula (I), R is a carbonate [-COOR _w ] selected from benzyl carbonate, p-nitrobenzyl carbonate, tert-butyl carbonate, 2,2,2- trichloroethyl carbonate, 2-(trimethylsilyl)ethyl carbonate, allyl carbonate.

It is noted that the following compounds, while can be used in the process of the invention, do not form part of the present invention as such:

- Estra-1 ,3,5(10)-triene-3, 15,16, 17-tetrol, 3-[4-(aminosulfonyl)benzoate], (15α,16α,17β);

- Estra-1 ,3,5(10)-triene-3, 15, 16, 17-tetrol, 3-[3-(aminosulfonyl)benzoate], (15α,16α,17β);

- Butanamide, 4-[[(15a, 16a, 17β)-15, 16,17-trihydroxyestra-1 ,3,5(10)-trien-3- yl]oxy];

- Butanoic acid, 4-[[(15a,16a,173)-15,16,17-trihydroxyestra-1 ,3,5(10)-trien-3- yl]oxy];

- Butanoic acid, 4-[[(15a,16a,173)-15,16,17-trihydroxyestra-1 ,3,5(10)-trien-3- yl]oxy], ethyl ester;

- β-D-Glucopyranosiduronic acid, (15a,16a,173)-15,16,17-trihydroxyestra- 1 ,3,5(10)-trien-3-yl, labeled with tritium;

- β-D-Glucopyranosiduronic acid, (15a,16a,173)-15,16,17-trihydroxyestra- 1 ,3,5(10)-trien-3-yl, monosodium salt;

- β-D-Glucopyranosiduronic acid, (15a,16a,173)-15,16,17-trihydroxyestra- 1 , 3,5(10)-trien-3-yl; and

- Estra-1 ,3,5(10)-triene-3, 15, 16, 17-tetrol, 3-(hydrogen sulfate), (15α,16α,17β)

Compounds of formula (II) of the invention

In another aspect the present invention refers to a compound of formula (II) per se

(II)

or a salt or solvate thereof,

wherein R represents an hydroxyl protecting group selected from:

- ethers [-R], where R can be selected from alkyl, cycloalkyi, aryl and arylalkyl, with the proviso that R is not C1 -C5 alkyl;

- alkoxy and aryloxy alkyl ethers such as alkoxy and aryloxy methyl ether [-CH ₂ - OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH ₂ -ORw], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyl;

- esters [-COR _w ], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyl, with the proviso that COR _w is not acetyl or C7-C12 benzoyl;

- amides [-CONR _w R _v ], where R _w and R _v can be independently selected from alkyl, cycloalkyi, aryl and arylalkyl; and

- carbonates [-COOR _w ], where R _w can be selected from alkyl, cycloalkyi, aryl and arylalkyl.

In a particular embodiment in the compound of formula (II), R is an ether [-R] selected from C6-alkyl; cycloalkyi such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; aryl such as phenyl, naphthyl, biphenyl, indenyl, phenanthryl; and arylalkyl such as benzyl, phenylethyl, phenylpropyl, naphthylmethyl.

In a particular embodiment in the compound of formula (II), R is a alkoxy or aryloxy alkyl ether such as alkoxy and aryloxy methyl ether [-CH ₂ -OR _w ] and alkoxy and aryloxy ethyl ether [-CH ₂ -CH ₂ -OR _w ] selected from methoxymethyl ether, 2- methoxyethoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, 2-(trimethylsilyl)ethoxymethyl ether, 1 -methoxyethyl ether, 1 -ethoxyethyl ether, 1 -n- propoxyethyl ether, 1 -isopropoxyethyl ether, 1 -n-butoxyethyl ether, 1 -isobutoxyethyl ether, 1 -sec-butoxyethyl ether, 1 -tert-butoxyethyl ether, 1 -ethoxy-n-propyl ether, methoxypropyl ether, ethoxypropyl ether, 1 -methoxy-1 -methylethyl ether, 1 -ethoxy-1 - methylethyl ether; tetrahydropyranyl and related ethers.

In a particular embodiment in the compound of formula (II), R is an ester [-COR _w ] selected from benzoyl, pivaloyl, methoxyacetyl, chloroacetyl, levulinyl ester.

In a particular embodiment in the compound of formula (II), R is an amide [-CONR _w R _v ] such as dimethylamide.

In a particular embodiment in the compound of formula (II), R is a carbonate [-COOR _w ] selected from benzyl carbonate, p-nitrobenzyl carbonate, tert-butyl carbonate, 2,2,2- trichloroethyl carbonate, 2-(trimethylsilyl)ethyl carbonate, allyl carbonate.

The following examples illustrate the invention and must not be considered in a limiting sense thereof.

Examples

Comparative Example 1 . Preparation of 3-benzyl-17-acetoxy-Estetrol (according to Example 9 of WO2004/041839)

A 5% suspension of PVP-Os0 ₄ (0.27 g/g) was prepared in 10 mL/g of THF. Then trimethylamine-N-oxide and a solution of 4.5 g of 3-benzyl-17-acetoxy-A-15-estradiol in 45 mL of THF were added at room temperature. It was heated at 50-55 °C until positive control. Cis-dihydroxylation products were measured in the HPLC control with a ratio of 80/20 (15α,16α / 15β,16β).

The reaction mixture was cooled to room temperature and the solid was filtered off, washed with THF (10 mL) and the organic layer was concentrated. The solid residue was dissolved in AcOEt (25 mL) and water (25 mL). 1 N aqueous HCI (1 .0 mL) was added to the aqueous layer. The two layers were separated and the aqueous layer was extracted with AcOEt (15 mL). Then, the organic layers were mixed and dried (Na ₂ S0 ₄ ). The organic phase was concentrated in vacuo until a residue was formed and then triturated with heptane/AcOEt (1 :1 ,10 mL) and stirred at room temperature. The solid obtained was filtered off to afford a solid (4.0 g, 88%). The product was purified by recrystallization from heptane/AcOEt/EtOH (2: 1 : 1 ) three times to afford a white solid (2.0 g, 44%).

Example 1 . Preparation of 3-benzoyl-Estrone

Over 30 g of Estrone in 300 mL of dichloromethane and 31 mL of triethylamine (d: 0.725 g/mL), 15.45 ml of benzoyl chloride were added at a temperature below 30 °C. After the addition, it was stirred at 20 ± 5 °C until positive control by TLC. Then, 120 mL of a solution of 10% V/V HCI was added, it was stirred and the two phases were separated. The aqueous phase was extracted with two aliquots of 60 mL of dichloromethane. The final organic phase was treated with 100 mL of 10% VWV of sodium bicarbonate and was extracted again with an aliquot of 60 mL of dichloromethane. The resulting organic phase was treated with 100 mL of water. This aqueous phase was also extracted with an aliquot of 60 mL of dichloromethane.

The final organic phase was concentrated under vacuum until a final volume of 90 mL. The solvent was changed with methanol by subsequent steps of addition and evaporation with three portions of 90 mL of methanol, concentrating in each case until a final volume of 90 mL. The final suspension was stirred at 0/5 °C for 30 minutes and then, the solid was filtered off. The solid was washed with 30 mL of methanol at 0/5 °C and it was dried at 50 °C, to afford a final dry cake: 42.5 g of 3-benzoyl-Estrone. Yield: 100% Molar.

Example 2. Preparation of 3-benzoyl-17,17-ethylenedioxy-Estrone

42 g of starting material 3-benzoyl-Estrone were suspended in 84 mL of ethylene glycol and 42 mL of triethylortoformiate and 0.84 g of p-toluenesulfonic acid were added. It was stirred at 35 ± 5 °C until positive control (about 15 hours). Then, 1.5 mL of pyridine were added at room temperature, and after stirring for 15 minutes, 600 mL of water were added over the mixture. The suspension was stirred at room temperature for 30 minutes and filtered. It was washed with 126 mL of water and dried at 50 °C. Final dry cake: 46.0 g of 3-benzoyl-17, 17-ethylenedioxy-Estrone. Yield: 98% Molar Example 3. Preparation of 3-benzoyl-16-bromo-17,17-ethylenedioxy-Estrone

A solution of 46.0 g of 3-benzoyl-17,17-ethylenedioxy-Estrone in 138 mL of THF and 9.2 mL of ethylene glycol was added over 45,5 g of pyridinium bromide in 42 mL of THF. The addition is performed maintaining the temperature at 20 ± 5 °C. After positive control (40 minutes) a solution of sodium triosulfate (0.3 g/mL) was added (this solution also can contain sodium carbonate). Then, dichloromethane (138 mL) was added and it was stirred at room temperature for 20 minutes. After separation, the solvent of the aqueous phase was changed by acetone with distillations and charges of acetone, obtaining a final suspension of 100 mL. It was stirred at 0/5 °C for 1 hour and filtered. It was washed with 23 mL of acetone and it was dried at 50 °C. Final dry cake: 49.8 g of 3-benzoyl-16-bromo-17,17-ethylenedioxy-Estrone. Yield: 91 % Molar.

Example 4. Preparation of A-15-17,17-ethylenedioxy-Estrone

Over 10 g of 3-benzoyl-16-bromo-17,17-ethylenedioxy-Estrone in 50 mL of DMSO, 15 g of potassium t-butoxide were added. The mixture was heated at 40/45°C for 1 hour. After positive control, the mixture was precipitated over water (200 mL) and the pH was adjusted with an aqueous solution of 10% sulphuric acid, to obtain a pH 7/8. The resulting suspension was filtered and washed with water (80 mL) to afford 13.5 g of Δ- 15-17,17-ethylenedioxy-Estrone as a wet cake.

The cake was drained and used as such in the next step. Example 5. Preparation of Δ-15-Estrone

The wet cake of the previous step A-15-17,17-ethylenedioxy-Estrone_was suspended again in 250 mL of acetone and 1 .5 g of p-toluenesulfonic acid were added (if pH is not below 3, more p-TSA is added). The reaction was complete after 1 hour at 20 ± 5 °C, and then 1 mL of pyridine was charged. The mixture was concentrated until 100 mL and 100 mL of water were added. It was filtered and washed with 50 mL of water.

The cake was drained and dried at 50 °C. Final dry cake: 32.51 g. Yield: 100 % Molar. Example 6. Preparation of Δ-15-Estradiol

Over 32.5 g of Δ-15-Estrone in 1 14 mL of THF, 488 mL of methanol and 9.75 g of cerium trichloride heptahydrate, 1 .4 g of sodium borohydride were added portion-wise at 0/5 °C. The reaction was complete in 10 minutes. Then 100 mL of water were added, it was concentrated under vacuum until 100 mL and 50 mL more of water are added. It was stirred at 5/10 °C for 1 hour, filtered and washed with 120 mL of water. The cake was drained and resuspended in methanol, filtered and dried at 50 °C. The final amount of dry cake is 30.5 g. Yield: 93 % Molar. Example 7. Preparation of Os0 supported on PVP [polv(4-vinyl-pyridine)l

1 gram of Osmium tetroxide was dissolved in 100 mL of cyclohexane and a suspension of 20 grams of PVP in 50 mL of cyclohexane was added under nitrogen atmosphere. It was stirred at 20 ± 5 °C for 18 hours. Then, it was filtered and washed with 200 mL of cyclohexane. It was dried in the filtration plate under vacuum.

The maximum amount of osmium tetroxide supported on PVP which can be obtained is 5% by weight.

Example 8. Preparation of Estetrol

Estetrol

Over 30.4 g of Estradiol in 335 mL of THF, and 6.0 g of PVP-Os0 ₄ , trimethylamine-N- oxide 2 H ₂ 0 (15.2 g) was added and then the mixture was heated at 55/60 °C. After 24 hours, it was filtered and the solid residue was extracted with THF. The liquids filtered were concentrated and it was charged with 100 mL of water, removing the solvent by distillation. The pH was adjusted at 7 with a solution of HCI and it was stirred at 20 ± 5 °C. It was filtered and washed with water. The cake was drained and dried at 50 °C. Final dry cake: 20.9 g. Yield: 62 % Molar.

The ratio between the 15a,16a-diol and 15β,16β-άίοΙ is 90/10. Example 9: Preparation of 3-Benzyl-A-15-Estradiol

H

Δ-15-Estradiol (10 g) was dissolved in a mixture of 40 ml of dichloromethane and 40 ml of Methanol under inert atmosphere, at room temperature. K ₂ C0 ₃ 1 .5 g were added and then 13.0 ml of Benzyl bromide were added thereto and the mixture was refluxed until completion of the reaction was observed by TLC. The mixture was cooled and the solid was filtered off and washed with Methanol (10 mL) Then, the organic phase was evaporated under reduced pressure. The suspended solid was filtered and washed with heptane and recrystallized in a mixture of dichloromethane/Methanol 1 :1 , filtered off and dried in an oven at 50°C to yield 12.01 g, 90% molar.

NMR-H ¹ :7.50-7.25 (5H); 7.12 (1 H); 6.72 (1 H); 6.68 (1 H); 5.94 (1 H); 5.63 (1 H); 5.00 (2H); 4.84 (1 H); 4.17 (1 H); 2.76 (2H); 2.23 (1 H); 2.13 (1 H); 2.00 (1 H); 1 .87 (2H); 1.60- 1 .2 (4H); 0.71 (3H).

NMR-C ¹³ : 156.1 ; 137.4; 135.9; 132.4; 129.9; 128.4; 127.6; 127.5; 125.9; 1 14.5; 1 12.24; 83.9; 68.9; 56.2; 50.88; 43.9; 40.1 ; 35.9; 34.5; 29.1 ; 27.1 ; 25.7; 12.5.

Example 10. Preparation of 3-Benzyl-Estetrol

3-benzyl-Estetrol

Over 12.7 g of 3-benzyl-A-15-Estradiol in 127.5 mL of THF, and 2.8 g de PVP-Os0 ₄ in 12.7 mL of THF, trimethylamine-N-oxide 2 H ₂ 0 (6.4 g) was added and then the mixture was heated at 55/60 °C. After about 24 hours, it was filtered and the solid residue was extracted with THF. The liquids filtered were concentrated and it was charged with 25 mL of water, removing the solvent by distillation. The pH was adjusted at 7 with a solution of HCI and it was stirred at 20 ± 5 °C. It was filtered and washed with water. The cake was drained and dried at 50 °C. Final dry cake: 13.8 g. Yield: 99 % Molar. The ratio between the 15a,16a-diol and 15β, 1 θβ-diol is 90/10.

NMR-H ¹ : 7.50-7.25 (5H); 7.14 (1 H); 6.73 (1 H); 6.68 (1 H); 5.04 (2H); 4.84 (1 H); 4.60 (1 H); 4.25 (1 H); 3.85-3.55 (2H); 3.20 (1 H); 2.75 (2H); 2.30-0.90 (9H); 0.67 (3H).

NMR-C ¹³ : 156.1 ; 137.6; 137.4; 132.3; 128.4; 127.6; 127.5; 126.3; 1 14.5; 1 12.24; 83.4; 75.0; 69.2; 69.0; 55.5; 43.7; 40.1 ; 39.1 ; 38.9; 29.5; 27.2; 25.7; 13.9.

Example 1 1 . Preparation of 3-Benzoyl-A-15-Estradiol

Δ-15-estradiol (10 g) was dissolved in 100 ml of dichloromethane and 1 1 ml of triethylamine under inert atmosphere, at a temperature of about 10-15°C and 5.15 ml of Benzoyl chloride were added thereto at a temperature lower than 30°C. The reaction mixture was stirred at room temperature for about one hour until completion of the reaction was observed by HPLC.

A 5% aqueous solution of hydrochloric acid (40 ml) was added at room temperature. The resulting mixture was kept until complete separation of the two phases. The aqueous phase was extracted twice with dichlormethane (20 ml x 2) and all the organic phases were mixed and washed with a 5% aqueous solution of NaHC0 ₃ (30 ml) and water (30 ml). After decantation, the aqueous phase is extracted with dichloromethane (20 ml). Then, the organic phases were mixed and evaporated under reduced pressure and ethyl acetate was added (30 ml x 3 times) and evaporated under reduced pressure. Heptane (60 ml) was added slowly to afford a suspension. The suspended solid was filtered and washed with heptane and dried in an oven at 50°C to yield 1 1.59 9-

NMR-H ¹ :8.08 (2H); 7.71 (1 H); 7.56 (2H); 7.29 (1 H); 6.97 (1 H); 6.93 (1 H); 5.95 (1 H); 5.56 (1 H); 4.84 (1 H); 4.18 (1 H); 2.83 (2H); 2.33-2.20 (2H); 2.05 (1 H); 1.90 (2H); 1 .65- 1 .25 (4H); 0.74 (3H).

NMR-C ¹³ : 164.7; 148.3; 137.8; 137.7; 136.0; 133.9; 129.9; 129.7; 129.0; 128.9; 126.1 ; 121 .5; 1 18.8; 83.36; 56.2; 50.9; 44.0; 35.7; 34.5; 28.8; 26.8; 25.6; 12.5. Example 12. Preparation of 3-Benzoyl-Estetrol

3-Benzoyl-A-15-Estradiol (10.1 g) was dissolved in 1 12 ml of THF under inert atmosphere. PVP-Os0 ₄ (2.0 g) and trimethylamine N-oxide 2 H ₂ 0 (5.1 g) were added at room temperature, the mixture was heated until 55-60°C for 20-24 h. Then, the mixture was filtered and the solid was extracted with THF. A first portion of the resulting organic phase was evaporated under vacuum, water (40 ml) was added to the liquid phase and the organic phase was evaporated under vacuum.

An aqueous solution of hydrochloric acid was added at room temperature to adjust the pH to 6.5-7.5. The mixture was stirred at room temperature for 1 -2 h. The suspension formed was filtered off and the solid washed with water (20 ml).

The wet cake was dried in an oven at 50°C to yield 10.1 g of Estetrol-3-benzoyl ester.

The ratio between the 15a,16a-diol and 15β,16β-άίοΙ is 90/10.

NMR-H ¹ : 8.08 (2H); 7.71 (1 H); 7.56 (2H); 7.29 (1 H); 6.97 (1 H); 6.93 (1 H); 4.84 (1 H); 4.61 (1 H); 4.24 (1 H); 3.75-3.50 (2H); 3.24 (1 H); 2.83 (2H); 2.33-0.90 (9H); 0.74 (3H).

NMR-C ¹³ : 164.7; 148.3; 137.8; 137.7; 129.9; 129.7; 128.9; 128.4; 126.1 ; 121 .5; 1 18.8;

83.36; 75.2; 69.2; 56.2; 44.0; 40.0; 35.7; 34.5; 28.8; 26.8; 25.6; 12.5.

Example 13. Preparation of 3-tert-Butyl-dimethyl-sylil-A-15-Estradiol

Δ-15-estradiol (3.0 g) was dissolved in 15 ml of THF under inert atmosphere at room temperature. Imidazol (2.0 g) and TBDMS chloride (3.18 g) were added. The reaction mixture was stirred at room temperature until completion of the reaction was observed by HPLC.

The resulting mixture was filtered off and the liquid phase was evaporated under reduced pressure and purified by column chromatography with the solvent (dichloromethane/ acetone/ triethylamine 99:1 :1 ) to yield 1.19 g of 3-tert butyl dimethyl silane-3, 173-Dihidroxi-A-15-estradiol. NMR-H ¹ : 7.07 (1 H); 6.54 (1 H); 6.49 (1 H); 5.90 (1 H); 5.60 (1 H); 4.81 (1 H); 4.16 (1 H); 2.75 (2H); 2.25 (1 H); 2.14 (1 H); 2.00 (1 H); 1 .87 (2H); 1 .60-1.20 (4H); 0.91 (9H); 0.71 (3H); 0.12 (6H).

NMR-C ¹³ : 152.7; 137.4; 136.0; 132.9; 129.9; 125.9; 1 19.5; 1 16.9; 83.9; 56.2; 50.9; 43.92; 36.9; 34.5; 28.8; 27.0; 25.8; 25.7; 25.6; 17.9; 12.5; -4.5.

Example 14. Preparation of 3-tert-Butyl-dimethyl-sylil-Estetrol

3-tert-butyl-dimethyl-sylil-A-15-Estradiol (1.0 g) was dissolved in 1 1 ml of THF under inert atmosphere. PVP-Os0 ₄ (0.2 g) and trimethylamine N-oxide (0.51 g) were added at room temperature, the mixture was heated until 55-60°C for 20-24 h. Then, the mixture was filtered and the solid was extracted with THF. A first portion of the resulting organic phase was evaporated under vacuum, water (4 ml) was added to the liquid phase and the organic phase was evaporated under vacuum.

An aqueous solution of hydrochloric acid was added at room temperature to adjust the pH to 6.5-7.5. The mixture was stirred at room temperature for 1 -2 h. The suspension formed was filtered off and the solid washed with water (2 ml).

The wet cake was dried in an oven at 50°C to yield 1 .1 g of 3-tert-butyl-dimethyl silane of Estetrol.

The ratio between the 15a,16a-diol and 15β,16β-άίοΙ is 90/10.

NMR-H ¹ : 7.07 (1 H); 6.54 (1 H); 6.49 (1 H); 4.81 (1 H); 4.59 (1 H); 4.16 (1 H); 3.85-3.55 (2H); 3.20 (1 H); 2.75 (2H); 2.25-1 .20 (9H); 0.91 (9H); 0.71 (3H); 0.12 (6H).

NMR-C ¹³ : 152.7; 137.4; 133.9; 125.9; 1 19.5; 1 16.9; 83.9; 76.8; 68.5; 56.2; 50.9; 41 .92; 36.9; 34.5; 28.8; 27.0; 25.8; 25.7; 25.6; 17.9; 12.5; -4.5.

Example 15. Preparation of 3-(1 -butoxyethyl) ether-Δ-15-estradiol.

Δ-15-Estradiol (2.0 g) was dissolved in 10 ml of THF and 1 .59 ml of n-butylvinyl ether under inert atmosphere at room temperature. Molecular sieves (0.2 g) and p- toluenesulphonic acid (0.2 g) were added. The reaction mixture was stirred at room temperature until completion of the reaction was observed by HPLC. Then pyridine was added and the resulting mixture was filtered off and the liquid phase was evaporated under reduced pressure and purified by column chromatography with the solvent (Ethyl acetate/ Heptane/ triethylamine 1 :10:1 ) to yield 2.01 g of 3-(1 -butoxyethyl) ether-A-15- estradiol.

Example 16. Preparation of 3-(1 -butoxvethyl) ether-Estetrol.

3-(1 -butoxyethyl) ether-A-15-estradiol (2.0 g) was dissolved in 22 ml of THF under inert atmosphere. PVP-Os0 ₄ (0.4 g) and trimethylamine N-oxide (1.0 g) were added at room temperature, the mixture was heated until 55-60°C for 20-24 h. Then, the mixture was filtered and the solid was extracted with THF. A first portion of the resulting organic phase was evaporated under vacuum, water (7.5 ml) was added to the liquid phase and the organic phase was evaporated under vacuum.

An aqueous solution of hydrochloric acid was added at room temperature to adjust the pH to 6.5-7.5. The mixture was stirred at room temperature for 1 -2 h. The suspension formed was filtered off and the solid washed with water (3.5 ml).

The wet cake was dried in an oven at 50°C to yield 2.1 g of 3-(1 -butoxyethyl) ether of

Estetrol.

The ratio between the 15a,16a-diol and 15β,16β-άίοΙ is 90/10.