PROCESS FOR THE PREPARATION OF (15α,16α,17β)-ESTRA-1,3,5(10)-TRIENE-3,15,16,17-TETROL (ESTETROL) AND ESTETROL MONOHYDRATE

Abstract

The present invention relates to a process for the preparation of (15α,16α,17β)-estra-1,3,5(10)-triene-3,15,16,17-tetrol, also known as estetrol, and estetrol monohydrate, having the formulas shown below:

Description

Title of the invention: PROCESS FOR THE PREPARATION OF (15a,16a,17P)-ESTRA-1,3,5(10)-TRIENE-3,15,16,17-TETROL (ESTETROL) AND ESTETROL MONOHYDRATE FIELD OF THE INVENTION

[0001] The present invention relates to the sector of processes for the synthesis of active ingredients for pharmaceutical use, and in particular to a process for the preparation on an industrial scale of the compound (15a,16a,17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol, also known as estetrol and in monohydrate form.

[0002] CONTEXT

[0003] The compound estetrol is an active ingredient exhibiting pharmacological activity that is useful in hormone replacement therapy (HRT), in female contraception, or in the therapy of autoimmune dysfunctions related to hormonal imbalances.

[0004] The structural formula of Testetrol is shown below:

[0005] [Chem.l] HCX''

[0006] estetrol

[0007] Positions 15, 16 and 17 of the steroid skeleton (specified in the formula presented above) each carry a hydroxyl which, as indicated in the structural formula, have a defined spatial arrangement.

[0008] Estetrol is a natural product isolated from human urine and has been known for years; it was described in the article "Synthesis of epimeric 15-hydroxyestriols, new and potential metabolites of estradiol", J. Fishman et al., JOC Vol. 33, No. 8, August 1968, pp. 3133-3135 (compound 1a in the figure on page 3133).

[0009] As for obtaining estetrol, the process obtainable from this article does not have industrial applicability due to the low yield of the process.

[0010] Several patent applications relating to new processes for the synthesis of estetrol have been published recently, but none avoid the formation of the 15[3,16[3,17[3] isomer, having the structural formula shown below, from which estetrol must be purified for use in pharmaceutical preparations.

[0011] [Chem.2]

[0012] isomer 15[3,16[3,17[3

[0013] For example, application WO 2004 / 041839 A2 (page 6, lines 5-10) describes a process for obtaining estetrol with a purity of up to 99%, with the sum of individual impurities not exceeding 1%. Example 11 on page 28 describes an estetrol with an HPLC purity of 99.1% (HPLC-Ms) which, however, does not provide information on the content of individual impurities; the limit accepted by international guidelines for pharmaceutical substances is 0.1% for unknowns and 0.15% for identified ones.

[0014] The content of impurities in an active ingredient (API) is an essential requirement that cannot be waived to enable its use in pharmaceutical preparations and is also a fundamental characteristic for defining an industrially applicable process. Any process, regardless of yield, providing an API with an impurity content that does not meet the limits of international guidelines is not an industrially useful process since the API, the result of the process, is not usable.

[0015] Subsequent applications relating to the production of estetrol are, for example, WO 2012 / 164096 A1, WO 2013 / 050553 A1 and WO 2015 / 040051 A1

[0016] In WO 2015 / 040051 A1 the estetrol / 15[3,16[3,17[3] isomer ratio is 99:1 in Examples 10 and 15, and 98:2 in Examples 11 and 17. In these examples, however, no indication is given to lower the content of the 15[3,16[3,17[3] isomer to at least 0.15%. Even chromatographic purification (Example 15) does not allow this result to be obtained. In this document it is noted (page 9, lines 5-15) that the methods described in the prior art discussed (represented in the case of this document by applications WO 2012 / 164096 A1 and WO 2013 / 050553 A1) provide even higher and unacceptable amounts of the 15[3,16[3,17[3 isomer.

[0017] It therefore appears evident that none of the methods described provides a solution to the limitation of the formation of the 15[3,16[3,17[3 isomer or a method for purifying estetrol from said isomer. Summary of the invention

[0018] The object of the present invention is to provide a method for synthesizing estetrol and of estetrol monohydrate having a content of the 15(3,16(3,17(3) isomer of less than 0.15%, without having to resort to purification techniques which are not industrially applicable.

[0019] The invention relates to a method for synthesizing estetrol which comprises the following steps:

[0020] A) oxidation of the compound (17(3)-3-(phenylmethoxy)-estra-l,3,5(10),15-tetraen-17-ol (intermediate 1) to give the compound (17(3)-3-(phenylmethoxy)-estra-l,3,5(10)-triene-15,16,17-triol (intermediate 2):

[0021] [Chem.3]

[0022] intermediate 1 intermediate 2

[0023] in which Bn = benzyl, and in which the configuration of carbon atoms 15 and 16 of the steroid skeleton of intermediate 2 is not fixed;

[0024] B) debenzylation of intermediate 2 to give the compound (17(3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 3) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is not fixed:

[0025] [Chem.4]

[0026] intermediate 2 intermediate 3

[0027] C) acetylation of intermediate 3 to tetraacetate of (17(3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 4) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is not fixed:

[0028] [Chem.5]

[0029] intermediate 3 intermediate 4

[0030] D) purification of intermediate 4 obtained in step C) into tetraacetate of (15a,16a,17(3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 5) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is fixed:

[0031] [Chem.6]

[0032] intermediate 4 intermediate 5

[0033] E) hydrolysis of the acetates present in intermediate 5 into estetrol:

[0034] [Chem.7]

[0035] intermediate 5 estetrol

[0036] F) reaction of the estetrol produced in step E) and transformation into estetrol monohydrate:

[0037] [Chem. 8]

[0038] estetrol estetrol monohydrate Brief description of the drawings

[0039] [Fig. 1] shows the HPLC chromatogram of estetrol monohydrate obtainable with the method of the invention.

[0040] [Fig.2] shows the DRX diffractogram of estetrol monohydrate obtainable with the method of the invention.

[0041] [Fig.3] shows the ACD curve of estetrol monohydrate obtainable with the process of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0042] The invention relates to a process for the synthesis of estetrol and estetrol monohydrate which comprises the steps defined above.

[0043] In the following description and in the claims, when the term "reduced pressure" is used, it denotes a pressure lower than 0.5 bar; when the term "until a low volume is obtained" is used in reference to an evaporation step, it denotes a residual volume of a solution lower than 50% of the initial volume.

[0044] Step A) of the process of the invention consists of the oxidation of the compound (17P)-3-(phenylmethoxy)-estra-l,3,5(10),15-tetraen-17-ol (intermediate 1) to give the compound (17[3]-3-(phenylmethoxy)-estra-l,3,5(10)-triene-15,16,17-triol (intermediate 2):

[0045] [Chem.9]

[0046] intermediate 1 intermediate 2

[0047] in which Bn = benzyl, and in which the configuration of carbon atoms 15 and 16 of the steroid skeleton of intermediate 2 is not fixed.

[0048] The starting substrate for this step, intermediate 1, can be obtained as described in application WO 2004 / 041839 A2.

[0049] As an oxidant in the reaction of step A) it is possible to use osmium tetroxide (OsO4) supported on a polymer or, preferably, as such, or potassium osmium dihydrate K2OsO4,2H2O. An organic amine N-oxide, such as trimethylamine N-oxide dihydrate, is used as a co-oxidant.

[0050] Since the oxidation with osmium derivatives is not stereoselective, intermediate 2 is obtained as a mixture of isomers having the configuration 15a,16a,17[3 and 15[3,16[3,17[3; the 15a,16a,17[3 isomer is produced in a predominant amount together with a minor amount of the 15[3,16[3,17[3 isomer.

[0051] The reaction is carried out in a solvent inert towards osmium derivatives, such as tetrahydrofuran (THF), at a temperature between 20 and 60°C, preferably between 30 and 50°C, and for a duration of at least 12 hours, preferably at least 16 hours.

[0052] The reaction can optionally be carried out in an inert atmosphere, preferably N2.

[0053] The reaction product (intermediate 2) after preparation can be treated with a product sequestering the metallic impurities in solution to remove the residual osmium content. These products, well known in chemistry, are generally based on a functionalized silica gel and commonly referred to in the sector by the term chemical trap, which will be used in the remainder of the text and the claims. The chemical trap is preferably QuadraSil® MP.

[0054] The treatment with the chemical trap can be carried out and can be repeated at each step of the process; it is preferably carried out in step F).

[0055] Step B) consists of the debenzylation of intermediate 2 to give the compound (17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 3) in which the confi- The configuration of carbon atoms 15 and 16 of the steroid skeleton is not fixed:

[0056] [Chem. 10]

[0057] intermediate 2 intermediate 3

[0058] Debenzylation consists of hydrogenation with hydrogen gas in the presence of a suitable catalyst. The preferred conditions for this reaction are:

[0059] - use of palladium on carbon (Pd / C) at 5% or 10% by weight, preference of palladium on carbon (Pd / C) at 5% by weight as catalyst;

[0060] - hydrogen pressure between 1 and 6 bar, preferably between 1 and 3 bar;

[0061] - a linear or branched C1 to C6 aliphatic alcohol, preferably methanol, in as a reaction solvent;

[0062] - reaction time of at least 12 hours, preferably at least 20 hours;

[0063] - hydrogenation temperature between 10 and 60°C, preferably between 15 and 55°C, of even more preferably between 20 and 50°C.

[0064] Step C) consists of the acetylation of intermediate 3 to tetraacetate of (17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 4) in which the confi The configuration of carbon atoms 15 and 16 of the steroid skeleton is not fixed:

[0065] [Chem. 11] OH r y-oh z V-OAC OAc HQ'" AcO •L .1 J oac

[0066] intermediate 3 intermediate 4

[0067] The complete acetylation of step C) is carried out in a solvent compatible with the reaction conditions, such as, for example, isopropyl acetate, ethyl acetate, tetrahydrofuran, pyridine or toluene. The preferred solvent is pyridine.

[0068] For the reaction, acetic anhydride is used as a feedstock, in an amount of at least 4, preferably 6 moles per mole of intermediate 3, in the presence of an inorganic or organic base and a catalyst.

[0069] Catalytic amounts of trifluoroacetic anhydride may be added.

[0070] Pyridine is preferably used as the organic base, and 4-dimethylaminopyridine (4-DMAP) as the catalyst.

[0071] The reaction temperature is between 5 and 40°C, preferably between 20 and 30°C; reaction time is at least 2 hours, preferably at least 3 hours.

[0072] The reaction can optionally be carried out in an N2 atmosphere.

[0073] Step D) consists of the purification of intermediate 4 obtained in step C) in te- (15a,16a,17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol traacetate (intermediate 5) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is fixed:

[0074] [Chem. 12] * OAc OAc ( I >'ÛAc ~ [ [ >"OAc £ XJ * X. XJ AcO AüO v

[0075] intermediate 4 intermediate 5

[0076] The purification of intermediate 4, with elimination of the 15[3,16[3,17[3] isomer, is obtained with the sequence of operations described below:

[0077] Dl) dissolving intermediate 4 to be purified in DCM at 15-30°C;

[0078] D.2) dropwise addition of the solution of intermediate 4 in DCM in pure methanol;

[0079] D.3) stirring the solution at 20-30°C for at least 10 minutes;

[0080] D.4) removal of the solvent by distillation under reduced pressure to obtain a suspension;

[0081] D.5) reflux of the suspension for at least 30';

[0082] D.6) cooling to 20-25°C and stirring for at least 1 h;

[0083] D.7) filtration of intermediate 5 and drying at reduced pressure for at least 3 h at 40-60°C.

[0084] The purification treatment can be repeated as many times as necessary to obtain the desired level of purity depending on the initial content of the 15[3,16[3,17[3 isomer.

[0085] The inventors carried out a series of experimental tests by repeating the sequence of operations Dl-D.7 several times on samples of intermediate 4 containing between 5 and 10% of 15[3,16[3,17[3 isomer to obtain a final product in which the content of the 15[3,16[3,17[3 isomer was less than 0.15% and in certain cases less than 0.05%.

[0086] Step E) consists of the hydrolysis of the acetates present in intermediate 5 into estetrol:

[0087] [Chem. 13]

[0088] intermediate 5 estetrol

[0089] The hydrolysis conditions are those known to those skilled in the art of organic chemistry.

[0090] The hydrolysis reaction of the acetates of intermediate 4 was carried out using bases in a solvent such as a linear or branched C1 to C6 aliphatic alcohol, or a mixture thereof, preferably methanol. The preferred conditions for this reaction are:

[0091] - use of sodium carbonate, potassium carbonate or potassium carbonate lithium as a base; preferably potassium carbonate is used;

[0092] - reaction time of at least 3 hours, preferably at least 4 hours;

[0093] - reaction temperature between 10 and 40 °C, preferably between 15 and 35 °C, of even more preferably between 20 and 30°C.

[0094] The solution containing the reaction product (estetrol) can optionally be:

[0095] • treated with a chemical trap based on functionalized silica gel to eliminate the residual palladium content; the chemical trap is preferably QuadraSil® MP; and / or

[0096] • purified by hot-cold crystallization in tetrahydrofuran (THF), methanol and acetonitrile, pure or as a mixture thereof.

[0097] In a second embodiment, the invention is focused on the preparation of estetrol in monohydrate form. In this embodiment, the method comprises an additional step, F),

[0098] [Chem. 14] » J / XL-t r T y "oh ' y "oh L il I ÔH k jl. J OH HO' HO'

[0099] estetrol estetrol monohydrate

[0100] which is carried out after step E) with the following sequence of operations:

[0101] Fl) dissolution of estetrol in a water-miscible organic solvent such as acetone, methanol, ethanol, isopropanol, tetrahydrofuran, dimethylformamide or dimethylacetamide until complete solution; the preferred solvent is methanol. In this operation, reflux heating may optionally be carried out to achieve a complete solution. The solution may optionally be treated with a functionalized silica gel chemical trap to remove residual palladium content. The chemical trap is preferably QuadraSil® MP. The solution may optionally be filtered through a Millipore membrane filter;

[0102] F.2) evaporation of the solution obtained in operation F. 1 under vacuum until obtaining a low volume;

[0103] F.3) addition of isopropyl alcohol (IPA), heating to 50-60 °C and evaporation under vacuum to a low volume. Step F.3 (addition of IPA and evaporation of the solvent) can be repeated as many times as necessary to obtain complete elimination of the solvent from step F1;

[0104] F.4) addition of isopropyl alcohol and heating to reflux (temperature su above 75°C) until the complete solution is obtained;

[0105] F.5) cooling the solution to 70-75°C;

[0106] F.6) addition of water (at least a volume equal to the volume of the organic solvent) and stirring at 60 < T < 70 °C;

[0107] F.7) removal of the IPA by distillation under reduced pressure at 55 < T < 65 °C;

[0108] F.8) cooling the suspension to 0 < T < 5 °C;

[0109] F.9) stirring at 0 < T < 5 °C for at least 30 minutes;

[0110] F. 10) filtration of the solid and drying at 30 < T < 50 °C for at least 16 h under reduced pressure. [YES] EXPERIMENTAL INSTRUMENTS, METHODS AND CONDITIONS

[0112] NMR:

[0113] JEOL 400 YH NMR spectrometer (400 MHz); JEOL Delta v5.1.1 software;

[0114] Spectra recorded in DMSO-d6.

[0115] SM:

[0116] Instrument: DSQ-trace Thermofisher

[0117] Sample introduction - direct exposure probe (sed)

[0118] Chemical ionization (CI) with methane

[0119] Methane pressure: 2.2 psi

[0120] Source temperature: 200°C

[0121] HPLC:

[0122] Agilent Model 1260 Infinity Chromatography System; UV Detector MODEL G1315C DAD VL+

[0123] HPLC Method 1:

[0124] Chromatographic conditions:

[0125] - Column: Supelco ascentis express C18 250 x 4.6 mm, 5 pm

[0126] - Flow rate: 1 ml / min

[0127] - Detector: UV 280 nm

[0128] - Injection volume: 5 pl

[0129] - Temperature: 25°C

[0130] - Mobile phase A: water

[0131] - Mobile phase B: acetonitrile

[0132] [Tables 1] TIME (min) MOVING PHASE A (v / v) MOVING PHASE B (v / v) 0 80 20 0-5 80 20 5-45 20 80 45-55 20 80 55-56 80 20 56-66 80 20

[0133] HPLC process 2:

[0134] Chromatographic conditions:

[0135] - Column: Supelco discovery Cl8 150 x 4.6 mm, 5 pm

[0136] - Flow rate: 1 ml / min

[0137] - Detector: UV 280 nm

[0138] - Injection volume: 25 μl

[0139] - Temperature: 22°C

[0140] - Mobile phase A: 4.29 g / l of CH3COONH4 solution in T water / methanol / acetonitrile 90 / 6 / 4

[0141] - Mobile phase B: 38.6 g / l of CH3COONH4 solution in 1 ' water / methanol / acetonitrile 10 / 5 4 / 3 6

[0142] [Tables2] TIME (min) MOVING PHASE A (v / v) MOVING PHASE B (v / v) 0 70 30 0-5 70 30 5-15 10 90 15-30 10 90 30-31 70 30 31-40 70 30

[0143] CLUP:

[0144] Waters Acquity UPLC; Detector: Acquity UPLC e X PDA Detector

[0145] CLUP process:

[0146] Chromatographic conditions:

[0147] - Column: Acquity UPLC BEH C18 1.7 pm, 2.1 x 50 mm

[0148] - Flow rate: 0.5 ml / min

[0149] - Detector: UV 225 nm

[0150] - Injection volume: 1 ft

[0151] - Temperature: 35°C

[0152] - Mobile phase A: water + 0.01% formic acid

[0153] - Mobile phase B: acetonitrile + 0.01% formic acid

[0154] [Tables3] TIME (min) MOVING PHASE A (v / v) MOVING PHASE B (v / v) 0 70 30 4-10 10 90 10-11 10 90 11-11.5 70 10 11.5-12 70 10

[0155] CLUP-MS System: Waters Acquity UPLC with Acquity UPLC PDA detector connected to a Waters Acquity UPLC QDa (ESI) detector

[0156] CCM:

[0157] MERCK: Aluminum foils for TLC 20 x 20 cm with F254 and silica gel 60, code 1.0554.0001.

[0158] CCM Detector:

[0159] Cerium phosphomolybdate: 25 g of phosphomolybdic acid and 10 g of cerium (IV) sulfate are dissolved in 600 ml of H2O. 60 ml of 98% H2SO4 are added and the resulting mixture is brought to 1 1 with H2O. The plate is impregnated with the solution and then heated until the products are detected.

[0160] DRXP:

[0161] XRPD analysis was performed using a Bruker D2 Phaser (2nd edition) powder diffractometer operating in Bragg-Brentano geometry, equipped with a rotating multi-sampler and a linear SSD detector (Lynxeye). The X-ray source is an X-ray tube with a copper anode operating at 30 kV and 10 mA. For the analysis, X-ray radiation with a wavelength corresponding to the average Ka of copper (X = 1.54184 Å) is used. The K[3 radiation is filtered through a nickel filter.

[0162] Flat surface silicone "zero background" sample holders were used on which the sample was spread to form a thin layer. During the analysis the sample holder is rotating at a speed of 60 rpm.

[0163] A scan is performed in the range 20 of 4 to 40° with increments 20 of 0.016° and an acquisition time of 1.0 s for each increment.

[0164] The diffractograms were processed using Bruker DIFFRAC.EVA software.

[0165] ACD:

[0166] The ACD analysis was carried out in an inert atmosphere (nitrogen) using a Perkin Elmer Diamond DSC differential scanning calorimeter. The samples were prepared by weighing the powder into 40 μl aluminum crucibles, which were then sealed before analysis. The analysis was carried out in the temperature range of 25 to 300 °C using a heating rate of 10 °C / min.

[0167] REMARKS

[0168] Water used in experimental descriptions is understood to be pure water unless otherwise indicated.

[0169] Organic solvents used in experimental descriptions are understood to be of “technical” quality, unless otherwise indicated.

[0170] The reagents and catalysts used in the experimental descriptions are understood to be of commercial quality, unless otherwise indicated.

[0171] QuadraSil® MP product is available from Johnson Matthey.

[0172] EXAMPLE 1

[0173] This example relates to step A) of the process of the invention, from intermediate 1 to intermediate 2.

[0174] [Chem. 15] OH CM

[0175] intermediate 1 intermediate 2

[0176] In a flask under nitrogen, 32.4 g of intermediate 1 (89.87 mmol, 1 eq) and 356 ml of tetrahydrofuran were charged. 0.324 g of osmium tetroxide (1.28 mmol, 1 wt%) and 17.9 g of trimethylamine N-oxide dihydrate (161.26 mmol, 1.8 eq) were added in this order to the solution. The system was heated to 50 °C and kept stirring for 16 hours.

[0177] The reaction was monitored by TLC analysis under the following conditions: TLC plate: silica gel on alumina; starting substrate (intermediate 1) dissolved in dichloromethane; reaction mixture diluted in dichloromethane; eluent: ethyl acetate (EtOAc); detector: cerium phosphomolybdate.

[0178] At the end of the reaction, the solution was cooled to 25 °C and a solution of sodium meta-bisulfite (18.3 g) in water (162 ml) was added dropwise. The solvent was concentrated under reduced pressure and 193 ml of isopropyl acetate and 290 ml of 1 M hydrochloric acid were added to the residue.

[0179] The phases were separated, and the aqueous phase was extracted with 160 ml of ethyl acetate. The organic solvent was washed with a solution of NaCl in water and the solution was added dropwise to 324 ml of pure n-heptane and stirred at 25 °C for 10 min (solution).

[0180] The ethyl acetate was removed under reduced pressure and the resulting suspension was stirred at 25°C for 1 h.

[0181] The solid was filtered through a Buchner filter washing with n-heptane and dried under reduced pressure at 50°C for 4 hours.

[0182] 30 g of intermediate 2 was obtained and used as is for the next step.

[0183] A small portion of intermediate 2 was purified for analytical purposes, to obtain the following data:

[0184] 'H-NMR (400 MHz, DMSO-d6): δ 7.31-7.43 (m, 5H); 7.15 (d, H, J = 8.8 Hz); 6.72-6.75 (m, 1H); 6.69 (s, 1H); 5.04 (s, 2H); 4.86 (d, H, J = 5.0 Hz); 4.61 (d, H, J = 6.0 Hz); 4.27 (d, H, J = 6.0 Hz); 3.67-3.73 (m, 2H); 3.25 (t, 1H); 2.74-2.77 (m, 2H); 1.03-2.22 (m, 9H); 0.67 (s, 3H).

[0185] Mass (ESI+): m / z = 395 [M++l], 377 [M++l-H20].

[0186] EXAMPLE 2

[0187] This example relates to step B) of the method of the invention.

[0188] [Chem. 16]

[0189] intermediate 2 intermediate 3

[0190] Intermediate 2 (8 g) obtained as described in the previous example was dissolved with 120 ml of methanol and charged into a hydrogenation reactor. 800 mg of 5% palladium on carbon was added to the solution and hydrogenation was carried out at 25 °C and 1 bar for 20 hours.

[0191] The reaction was monitored by TLC analysis under the following conditions: TLC plate: silica gel on alumina; starting substrate (intermediate 2) dissolved in dichloromethane (DCM); reaction mixture diluted with methanol (MeOH); eluent: DCM / MeOH 9 / 1; detector: cerium phosphomolybdate. At the end of the reaction, the system was filtered through a layer of dicalite, washing with methanol.

[0192] The solvent was concentrated under reduced pressure to a residual volume of 20 ml and 60 ml of water was added (precipitation of solid was detected).

[0193] The suspension was concentrated under reduced pressure to remove residual methanol.

[0194] The suspension was stirred for 30 minutes at 20-25 °C. The solid was filtered through a Buchner filter by washing with water and dried under reduced pressure at 50°C for 6 hours.

[0195] 5.65 g of intermediate 3 (white solid) were obtained.

[0196] The amount of 15[3,16[3,17[3] isomer present in the reaction product was determined by HPLC analysis and represents 7.8% of the desired 15a,16a,17[3] isomer.

[0197] EXAMPLE 3

[0198] This example relates to the implementation of step C) of the method of the invention.

[0199] [Chem. 17]

[0200] intermediate 3 intermediate 4

[0201] 5 g of intermediate 3 obtained as described in the previous example and 35 ml of pyridine were loaded into a flask and shaken at 20-25°C (clear solution).

[0202] 160 mg of 4-dimethylamino pyridine (4-DMAP) was added to the solution and, After 10' of stirring at 20-25°C, 9.3 ml of acetic anhydride was added.

[0203] The reaction was monitored after 4 h of stirring at 25 °C by TLC analysis under the following conditions: TLC plate: silica gel on alumina; starting substrate (intermediate 3) dissolved in dichloromethane; reaction mixture diluted with 1 M HCl and ethyl acetate (EtOAc); eluent: heptane / EtOAc 2 / 8; detector: cerium phosphomolybdate.

[0204] At the end of the reaction the solvent was concentrated at reduced pressure and 35 ml of DCM and 18 ml of water were added.

[0205] The two-phase system was kept stirring at 25°C for 10' then neutralized with 12 M hydrochloric acid while cooling, and stirred for 30 minutes.

[0206] The organic solvent of the biphasic system was washed with an aqueous solution of NaHCO3 followed by washing with water and, finally, washing with an aqueous solution of NaCl.

[0207] The solvent was completely removed by distillation under reduced pressure to obtain 7.4 g of crude intermediate 4 (solid).

[0208] The 15[3,16[3,17[3] isomer present in crude intermediate 4 was determined by HPLC analysis (method 1) and represents 7.5% of the desired 15a,16a,17[3] isomer.

[0209] EXAMPLE 4

[0210] This example relates to the implementation of step D) of the method of the invention.

[0211] [Chem. 18]

[0212] intermediate 4 intermediate 5

[0213] Crude intermediate 4 (7.4 g), obtained as described in the previous example, was dissolved in 29.6 ml of DCM (solution).

[0214] The solution of intermediate 4 in DCM was added dropwise to 74 ml of pure methanol and stirred at 25 °C for 10' (solution).

[0215] The organic solution was concentrated at 45 °C under reduced pressure to a final volume of 44 ml (suspension).

[0216] The suspension was brought to reflux (65°C) for 30' (suspension) then cooled to 25°C with stirring for at least 1 h.

[0217] The solid was filtered through a Buchner filter, washing with methanol, and dried under reduced pressure for 3 hours at 45°C.

[0218] Crude intermediate 4 (5.5 g; isomer 15[3,16[3,17[3 = 0.17%) was dissolved in 22 ml of DCM (solution).

[0219] The DCM solution of intermediate 4 was added dropwise to 55 ml of pure methanol and stirred at 25 °C for 10' (solution).

[0220] The organic solution was concentrated at 45°C under reduced pressure to a final volume of 44 ml (suspension).

[0221] The suspension was brought to reflux (65°C) for 30' (suspension) then cooled to 25°C with stirring for at least 1 h.

[0222] The solid was filtered through a Buchner filter, washing with methanol, and dried under reduced pressure for 3 hours at 45°C.

[0223] 5.2 g of pure intermediate 5 was obtained, which was analyzed by HPLC.

[0224] Pure intermediate 5 had HPLC purity (method 1) = 98.9%, with the isomer 15[3,16[3,17[3 not detectable.

[0225] 'H-NMR (400 MHz, CDC13): δ 7.27-7.26 (m, 1H); 6.83-7.86 (m, 1H); 6.79 (s, 1H); 5.39 (t, 1H); 5.16 (t, 1H); 5.01 (d, H, J = 6.4 Hz); 2.83-2.87 (m, 2H); 2.28-2.23 (m, 2H); 2.28 (s, 3H); 2.09 (s, 3H); 2.05 (s, 6H); 1.50-1.85 (m, 7H); 0.94 (s, 3H).

[0226] Mass (ESI+): m / z = 473 [M++l], 413 [M++l-AcOH], 353 [M++l-2AcOH], 293 [M +l-3AcOH],

[0227] EXAMPLE 5

[0228] This example relates to the implementation of step E) of the method of the invention.

[0229] [Chem. 18]

[0230] intermediate 5 estetrol

[0231] 2.5 g of the intermediate obtained in Example 4 were dissolved in 50 ml of methanol and 650 mg of potassium carbonate were added.

[0232] The mixture was kept stirring at 25°C for 4 hours (solution).

[0233] The reaction was monitored by TLC analysis under the following conditions: TLC plate: silica gel on alumina; intermediate 5 dissolved in dichloromethane; reaction mixture quenched in 1 M HCl and extracted with EtOAc, the organic phase was deposited; eluent: heptane / EtOAc 2 / 8; detector: cerium phosphomolybdate.

[0234] The solution was concentrated under reduced pressure to a residual volume of 5 ml, 18.5 ml of water was added, and the residual methanol was removed under reduced pressure.

[0235] The resulting suspension was neutralized with 1 M hydrochloric acid (pH ~ 7) and cooled to 10 °C while stirring for 60 minutes. The solid was filtered through a Buchner filter washing with water and dried under reduced pressure at 50 °C for 6 hours.

[0236] 1.5 g of estetrol (white solid) was obtained.

[0237] HPLC purity (method 2) = 99.4%, 15[3,16[3,17[3 isomer not detectable.

[0238] Mass (ESI+): m / z = 305 [M++l], 287 [M++l-H20], 269 [M++1-2H2O], 251 [M+ +1-3H2O].

[0239] EXAMPLE 6

[0240] This example relates to the implementation of step F) of the method of the invention.

[0241] [Chem. 19]

[0242] estetrol estetrol monohydrate

[0243] 3.5 g of estetrol obtained by following the experimental procedures described in the previous examples were suspended with stirring in 63 ml of methanol.

[0244] The suspension was heated to reflux temperature until completely dissolved.

[0245] The solution was cooled to 20-25°C and QuadraSil® MP was added while keeping the solution stirring for 16 h.

[0246] The thick suspension (estetrol solution - methanol and QuadraSil® MP) was filtered.

[0247] The solution (estetrol and methanol) was warmed to 45°C and filtered through a Millipore membrane filter, washing with MeOH.

[0248] The solution was evaporated under reduced pressure and 14 ml of isopropyl alcohol (IPA) was added to the residual volume of 28 ml, maintaining T > 50 °C.

[0249] This last step was repeated two more times (final volume 28 ml).

[0250] Finally, 17.5 ml of IPA was added and the whole was brought to reflux until complete dissolution of solids.

[0251] The solution was cooled to T = 70 °C and 45.5 ml of water was added while maintaining T > 60 °C under stirring.

[0252] Slowly the suspension was distilled while maintaining T = 55-65 °C under reduced pressure to a residual volume of 35 ml.

[0253] The thick suspension was slowly cooled to 5°C, stirred for at least 30 minutes at this temperature and filtered through a Buchner filter.

[0254] The filter cake was washed with water and the solid was dried in a vacuum oven at 35°C for about 18 h.

[0255] Estetrol monohydrate (white solid) was obtained (3.50 g; KF = 5.72%).

[0256] Estetrol monohydrate was analyzed by HPLC (method 2).

[0257] The test results are shown in [Fig.l]: the product was found to be estetrol monohydrate of HPLC purity = 100%.

[0258] 'H-NMR (400 MHz, DMSO-d6): δ 9.0 (s, 1H); 7.05 (d, H, J = 8.4 Hz); 6.51-6.48 (m, 1H); 6.41 (d, H, J = 2.4 Hz); 4.85 (d, H, J = 4.8 Hz); 4.60 (d, H, J = 5.6 Hz); 4.27 (d, H, J = 6 Hz); 3.66-3.71 (m, 2H); 3.23-3.26 (t, 1H, J = 5.6 Hz); 2.68-2.73 (m, 2H); 2.18-2.22 (m, 2H); 2.05-2.10 (m, 1H); 1.73-1.76 (d, H, 12Hz); 1.02-1.45 (m, 5H); 0.66 (s, 3H).

[0259] Mass (ESI+): m / z = 305 [M++l], 287 [M++l-H20], 269 [M++1-2H2O], 251 [M+ +1-3H2O].

[0260] A sample of the product was subjected to DRXP analysis; the result of the test is the diffractogram shown in [Fig.2].

[0261] The list below indicates the positions (as angle values 20 ± 0.2°) and relative intensities of the main peaks of the diffractogram:

[0262] 6,889, 28.6%; 12,087, 13.2%; 12,567, 60.7%; 13,259, 8.7%; 13,619, 63.3%; 14,983, 6.6%; 17,533, 10.6%; 18,621, 9.1%; 20,871, 100.0%; 21,757, 11.8%; 23,139, 17.2%; 25,399, 7.2%; 30,736, 6.8%; 34,642, 6.0%; 38,359, 7.9%.

[0263] Another sample of the product obtained, weighing 3.4 mg, was subjected to an ACD test; the result of the test is shown in [Fig. 3], which shows a first broadened peak attributed to the dehydration of estetrol monohydrate, and a second peak at about 244-245 °C, namely at a temperature essentially corresponding to the melting temperature of estetrol.

Claims

1. Claims A process for the synthesis of estetrol and estetrol monohydrate comprising the following steps: • A) oxidation of the compound (17[3]-3-(phenylmethoxy)-estra-1,3,5(10),15-tetraen-17-ol (intermediate 1) to give the compound (17[3)-3-(phenylmethoxy)-estra-l,3,5(10)-triene-15,16,17-triol (intermediate 2): intermediate 1 intermediate 2 • in which Bn = benzyl, and in which the configuration of carbon atoms 15 and 16 of the steroid skeleton of intermediate 2 is not fixed; • B) Debenzylation of intermediate 2 to give the compound (17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 3) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is not fixed: intermediate 2 intermediate 3 • C) Acetylation of intermediate 3 to tetraacetate of (17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 4) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is not fixed: OA& intermediate 3 intermediate 4 D) purification of intermediate 4 obtained in step C) into tetraacetate of (15a,16a,17[3)-estra-l,3,5(10)-triene-3,15,16,17-tetrol (intermediate 5) in which the configuration of carbon atoms 15 and 16 of the steroid skeleton is fixed: AcO" OÀc >æOAc bAc- OAc / ■"OAc ÔAC intermediate 4 intermediate 5 • E) Hydrolysis of the acetates present in intermediate 5 into estetrol intermediate 5 estetrol • F) The estetrol produced in step E) is transformed into estetrol monohydrate h2o

2. estetrol estetrol monohydrate. The method of claim 1, wherein step A) is carried out using an osmium compound as such or supported on a polymer in as oxidant and an organic amine N-oxide as cooxidant, operating in a solvent inert towards osmium derivatives, at a temperature between 20 and 60°C, and for a duration of at least 12 hours.

3. A process according to any one of the preceding claims, wherein step B), the debenzylation reaction, is carried out under the following conditions: - use of palladium on carbon (Pd / C) at 5% or 10% by weight as catalyst; - hydrogen pressure between 1 and 3 bar; - a linear or branched C1 to C6 aliphatic alcohol as reaction solvent; - reaction time of at least 12 hours; - hydrogenation temperature between 10 and 60 °C.

4. A process according to any one of the preceding claims wherein step C), the complete acetylation reaction of intermediate 3 to intermediate 4, is carried out using acetic anhydride as feed in a solvent selected from isopropyl acetate, ethyl acetate, tetrahydrofuran, pyridine and toluene, in the presence of an inorganic or organic base, with trifluoroacetic anhydride or 4-dimethylaminopyridine (4-DMAP) as catalyst and operating at a temperature between 5 and 40°C for a period of at least 2 hours.

5. A process according to any one of the preceding claims wherein step D), the purification of intermediate 4 to give intermediate 5, is carried out with the following sequence of operations: D1) dissolving intermediate 4 to be purified in DCM at 15-30°C; D.2) adding the solution of intermediate 4 in DCM dropwise into pure methanol; D.3) stirring the solution from operation D.2) at 20-30°C for at least 10 minutes; D.4) removing the solvent by distillation under reduced pressure to obtain a suspension; D.5) refluxing the suspension for at least 30' (suspension); D.6) cooling the suspension to 20-25°C and stirring for at least 1 h; D.7) filtering intermediate 5 and drying at reduced pressure for at least 3 hours at 40-60°C.

6. A method according to any one of the preceding claims, wherein the purification step D) is repeated the number of times necessary to obtain the desired level of purity according to the initial content of the 15[3,16[3,17[3 isomer, wherein said desired level of purity corresponds to a content of the 15[3,16[3,17[3 isomer < 0.15%.

7. Process according to any one of the preceding claims, in which the hydrolysis reaction of step E), of intermediate 5 to estetrol, is carried out under the following conditions: - use of sodium carbonate, potassium carbonate or lithium carbonate as base in a solvent chosen from linear or branched C1 to C6 aliphatic alcohols, or a mixture thereof; - reaction time of at least 3 hours; - reaction temperature between 10 and 40 °C.

8. A process according to any one of the preceding claims, wherein in step F) estetrol is converted into estetrol monohydrate with the following sequence of operations: F1) dissolving estetrol in a water-miscible organic solvent; F.2) evaporating the organic solvent under vacuum to a low volume; F.3) adding isopropyl alcohol (IPA), heating to 50-60°C and evaporating the organic solvent under vacuum to a low volume; F.4) adding isopropyl alcohol and heating under reflux until the complete solution is obtained; F.5) cooling the solution to 70-75°C; F.6) adding water and stirring at 60 < T < 70°C; F.7) removing PIPA by distillation under reduced pressure at 55 < T < 65°C to obtain a suspension; F.8) cooling the suspension to 0 < T < 5 °C; F.9) stirring at 0 < T < 5 °C for at least 30 minutes; F.10) filtration of the solid and drying at 30 < T < 50 °C for at least 16 h under reduced pressure.

9. A method according to any preceding claim, wherein the solution from step F1) is refluxed until a complete solution is reached.

10. A method according to claim 9, wherein said solution is treated with a functionalized silica gel-based chemical trap to remove residual palladium content.

11. The method of claim 10, wherein said chemical trap is QuadraSil® MP.

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